Treating neural disease with tyrosine kinase inhibitors

ABSTRACT

Provided herein are methods of treating or preventing a neurodegenerative disease, a myodegenerative disease or a prion disease in a subject comprising administering a tyrosine kinase inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 14/398,379, filed Oct.31, 2014, which is a U.S. national stage application under 35 U.S.C. §371 of PCT/US2013/039283, filed May 2, 2013, which claims the benefit ofU.S. Provisional Application No. 61/641,441, filed May 2, 2012, and U.S.Provisional Application No. 61/771,515, filed Mar. 1, 2013. Theabove-listed applications are hereby incorporated herein by thisreference in their entireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numberAG30378 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND

Neurodegenerative diseases include genetic and sporadic disordersassociated with progressive nervous system dysfunction. It has beenestimated that one of four Americans will develop a neurodegenerativecondition in their lifetimes. Generally, however, the underlyingmechanisms causing the conditions are not well understood and feweffective treatment options are available for preventing or treatingneurodegenerative diseases. Similarly, treatment options formyodegenerative disease and prion disease are also limited.

SUMMARY

Provided herein is a method of treating or preventing aneurodegenerative disease, a myodegenerative disease or a prion diseasein a subject, comprising selecting a subject with a neurodegenerativedisease of the central nervous system, a myodegenerative disease or aprion disease or at risk for a neurodegenerative disease of the centralnervous system, a myodegenerative disease or a prion disease andadministering to the subject an effective amount of a tyrosine kinaseinhibitor, wherein the tyrosine kinase inhibitor is not Gleevec, andwherein the tyrosine kinase inhibitor crosses the blood brain barrier.

Further provided is a method of inhibiting or preventing toxic proteinaggregation in a neuron, a muscle cell or a glial cell comprisingcontacting the neuron, the muscle cell or the glial cell with aneffective amount of a tyrosine kinase inhibitor, wherein the tyrosinekinase inhibitor is not Gleevec and wherein the tyrosine kinaseinhibitor crosses the blood brain barrier.

Also provided is a method of rescuing a neuron from neurodegeneration, amuscle from myodegeneration or a glial cell from degeneration comprisingcontacting the neuron, the muscle cell or the glial cell with aneffective amount of a tyrosine kinase inhibitor, wherein the tyrosinekinase inhibitor is not Gleevec and wherein the tyrosine kinaseinhibitor crosses the blood brain barrier.

Further provided herein is a method of treating amyotrophic lateralsclerosis or frontotemporal dementia in a subject, comprising selectinga subject with amyotrophic lateral sclerosis or frontotemporal dementia,wherein the subject has a TDP-43 pathology, and administering to thesubject an effective amount of a tyrosine kinase inhibitor, wherein thetyrosine kinase inhibitor is not Gleevec and wherein the tyrosine kinaseinhibitor crosses the blood brain barrier.

Also provided is a method of promoting parkin activity in a subject,comprising selecting a subject with a disorder associated with decreasedParkin activity and administering to the subject an effective amount ofa small molecule that increase parkin activity, wherein the smallmolecule is not Gleevec.

Further provided is a method of treating or preventing aneurodegenerative disease in a subject, comprising selecting a subjectwith a neurodegenerative disease or at risk for a neurodegenerativedisease, determining that the subject has a decreased level of parkinactivity relative to a control, and administering to the subject aneffective amount of a small molecule that increases parkin activity,wherein the small molecule is not Gleevec.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing the cellular mechanisms associated withparkin activity in neurodegenerative conditions (left) and uponintervention with tyrosine kinase inhibitors (right). Interventionactivates parkin activity to promote clearance of autophagic vacuoles.

FIG. 2 is a diagram showing that amyloid accumulation leads toautophagic induction and sequestration in phagophores. In transgenic oramyloid expressing animals parkin interaction with beclin-1 is reduced,leading to decreased maturation of phagophore into autophagosomes andautophagic defects. Kinase inhibition activates parkin and increases itsinteraction with beclin-1, resulting in maturation of phagophores intophagosomes and clearance. Subcellular fractionation via metrazimidegradients to isolate the phagophore (AV-10), autophagosomes (AV-20) andthe lysosomes was used to show how the cell handles amyloid accumulationand clearance.

FIG. 3 shows that parkin interacts with beclin-1 in wild type but notparkin−/− mice: Proximity Ligation Assay (PLA) in situ on 20 mm thickbrain sections showed parkin and beclin-1 interaction in A) C57BL/6 micebut not B) parkin−/− mice (control), indicating that parkin interactswith beclin-1. PLA in situ on 20 mm thick brain sections showed parkinand beclin-1 interaction in C) Tg-A53T and D) Tg-APP mice treated withDMSO, E) Tg-A53T and F) Tg-APP treated with 10 mg/kg nilotinib for 3weeks, G) Tg-A53T and H) Tg-APP treated with 5 mg/kg bosutinib for 3weeks.

FIG. 4 is a graph representing ELISA levels of human Aβ₁₋₄₂ in brainlysates of triple mutant APP-AD mice (Tg-APP) treated with either 1mg/kg or 5 mg/kg Nilotinib once every two days for 6 weeks. N=10animals. P<0.05. ANOVA, with Neuman Keuls multiple comparison. Anasterisk indicates a significant difference compared to DMSO. Bars aremean±SD.

FIG. 5 is a graph representing ELISA levels of human Aβ₁₋₄₂ in brainlysates of triple mutant APP-AD mice (Tg-APP) treated with either 1mg/kg or 5 mg/kg bosutinib once every two days for 6 weeks. N=10animals. P<0.05. ANOVA With Neuman Keuls multiple comparison. Anasterisk indicates a significant difference as compared to DMSO. Barsare mean±SD.

FIG. 6 is a graph representing ELISA levels of human α-synuclein inbrain lysates of A53T mice (A53T-Tg) treated with 5 mg/kg Bosutinib oncea day for 3 weeks. N=10 animals. P<0.05. ANOVA, with Neuman Keulsmultiple comparison. An asterisk indicates a significant difference ascompared to DMSO. Bars are mean±SD.

FIG. 7 is a graph representing ELISA levels of human α-synuclein inbrain lysates of A53T mice (A53T-Tg) treated with either 1 mg/kg or 5mg/kg Bosutinib once every 2 days for 6 weeks. N=10 animals. P<0.05.ANOVA, with Neuman Keuls multiple comparison. An asterisk indicates asignificant difference as compared to DMSO. Bars are mean±SD.

FIG. 8 is a graph representing ELISA levels of human α-synuclein inblood of A53T mice (A53T-Tg) treated with either 1 mg/kg or 5 mg/kgBosutinib once every 2 days for 6 weeks. N=10 animals. P<0.05. ANOVA,with Neuman Keuls multiple comparison. An asterisk indicates asignificant difference as compared to DMSO. Bars are mean±SD.

FIG. 9 is a graph representing ELISA levels of human α-synuclein inbrain lysates of A53T mice (A53T-Tg) treated with either 1 mg/kg or 5mg/kg Nilotinib once every second day for 6 weeks. N=10 animals. P<0.05.ANOVA, with Neuman Keuls multiple comparison. An asterisk indicates asignificant difference as compared to DMSO. Bars are mean±SD.

FIG. 10 is a graph representing ELISA levels of human α-synuclein inblood of A53T mice (A53T-Tg) treated with either 1 mg/kg or 5 mg/kgNilotinib once every second day for 6 weeks. N=10 animals. P<0.05.ANOVA, with Neuman Keuls multiple comparison. An asterisk indicates asignificant difference as compared to DMSO. Bars are mean±SD.

FIG. 11 shows A) a graph representing ELISA levels of human Aβ1-42, B) agraph representing human Aβ1-40 in brain lysates of triple mutant APP-ADmice (Tg-APP) treated with 5 mg/kg Bosutinib every day for 3 weeks, C) agraph representing ELISA levels of mouse parkin and D) a graphrepresenting mouse phosphorylated Tau (Ser 396) in brain lysates oftriple mutant APP-AD mice (Tg-APP) treated with 5 mg/kg Bosutinib everyday for 3 weeks. N=10 animals. P<0.05. ANOVA With Neuman Keuls multiplecomparison. An asterisk indicates a significant difference as comparedto DMSO. Bars are mean±SD.

FIG. 12 is a graph representing ELISA levels of human Aβ₁₋₄₂ in brainlysates of lentiviral Aβ₁₋₄₂ injected mice (wild type and parkin_(−/−)for 3 weeks and treated with 5 mg/kg Bosutinib every day for 3additional weeks. N=10 animals. P<0.05. ANOVA with Neuman Keuls multiplecomparison. An asterisk indicates a significant difference as comparedto DMSO. Bars are mean±SD.

FIG. 13 shows that α-synuclein expression in the brain increases itsblood level and tyrosine kinase inhibition reverses these effects in aparkin-dependent manner. Mice were injected stereotaxically(bilaterally) with lentiviral α-synuclein into the substantia nigra for3 weeks. Then, half of the animals were injected with 10 mg/Kg nilotiniband the other half with DMSO. The effects of α-synuclein expression andtyrosine kinase inhibition on A) brain and B) blood levels ofα-synuclein were compared. An asterisk indicates a significantdifference as compared to DMSO. Bars are mean±SD.

FIG. 14 shows that α-synuclein expression in the brain increases itsblood level and tyrosine kinase inhibition reverses these effects in aparkin-dependent manner. Mice were injected stereotaxically(bilaterally) with lentiviral α-synuclein into the substantia nigra for3 weeks. Then, half of the animals were injected with 5 mg/Kg bosutiniband the other half with DMSO. The effects of α-synuclein expression andtyrosine kinase inhibition on A) brain and B) blood levels ofα-synuclein were compared. An asterisk indicates a significantdifference as compared to DMSO. Bars are mean±SD.

FIG. 15 shows that α-synuclein induced loss of dopamine and homovanillicacid (HVA) levels. Tyrosine kinase inhibition reversed these effects andimproved motor performance. Mice were injected stereotaxically(bilaterally) with lentiviral α-synuclein into the substantia nigra for3 weeks. Then, half the animals were injected with 10 mg/kg Nilotinib or5 mg/Kg Bosutinib and the other half with DMSO. The effects ofα-synuclein expression and tyrosine kinase inhibition on A) dopamine andhomovanillic acid (HVA) levels (ELISA) were compared. The effects oftreatment on B) motor performance were evaluated using rotarod. Anasterisk indicates a significant difference as compared to DMSO. Barsare mean±SD.

FIG. 16 shows that Aβ₁₋₄₂ accumulates in AV-10 in Tg-APP animals butdrug treatment enhances autophagic clearance via deposition of Aβ₁₋₄₂ inAV-20 and lysosome. Histograms show Aβ₁₋₄₂ in subcellular fractions,including autophagic vacuole-10 (AV-10; phagophores+autophagosomes),AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice wereinjected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once aday for 3 consecutive weeks. Brain tissues were fractionated to isolateAVs and human specific ELISA was performed to determine proteincontents. N=5 animals per treatment.

FIG. 17 shows that Aβ₁₋₄₀ accumulates in AV-20 in Tg-APP animals butdrug treatment enhances autophagic clearance via deposition of Aβ₁₋₄₀ inAV-20 and lysosome. Histograms show Aβ₁₋₄₀ in subcellular fractions,including autophagic vacuole-10 (AV-10; phagophores+autophagosomes),AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice wereinjected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once aday for 3 consecutive weeks. Brain tissues were fractionated to isolateAVs and specific ELISA was performed to determine protein contents. N=5animals per treatment.

FIG. 18 shows that P-Tau accumulates in AV-10 in Tg-APP animals but drugtreatment enhances autophagic clearance via deposition of p-Tau in AV-20and lysosome, which contains degradative enzymes. Histograms show Tauhyper-phosphorylation (p-Tau) at serine 396 in subcellular fractions,including autophagic vacuole-10 (AV-10; phagophores+autophagosomes),AV-20 (autophagosomes) and lysosomes. Transgenic 3×APP mice wereinjected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once aday for 3 consecutive weeks. Brain tissues were fractionated to isolateAVs and mouse-specific ELISA was performed to determine proteincontents. N=5 animals per treatment.

FIG. 19 shows that drug treatment increases parkin activity leading toprotein clearance including parkin itself. Histograms show parkin insubcellular fractions, including autophagic vacuole-10 (AV-10;phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes.Transgenic 3×APP mice were injected IP with 10 mg/kg Nilotinib or 5mg/kg Bosutinib or DMSO once a day for 3 consecutive weeks. Braintissues were fractionated to isolate AVs and mouse specific ELISA wasperformed to determine protein contents. Parkin accumulates in AV-10 inTg-APP animals but drug treatment enhances autophagic clearance viadeposition of parkin in AV-20 and lysosome, which contains degradativeenzymes. N=5 animals per treatment.

FIG. 20 shows that autophagic clearance is parkin-dependent. Histogramsshow Aβ₁₋₄₂ in subcellular fractions, including autophagic vacuole-10(AV-10; phagophores+autophagosomes), AV-20 (autophagosomes) andlysosomes. Wild type or parkin−/− mice were injected with lentiviralAβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/KgBosutinib or DMSO once a day for 3 (additional) consecutive weeks. Braintissues were fractionated to isolate AVs and human specific ELISA wasperformed to determine protein contents. Aβ₁₋₄₂ accumulates in AV-10 inlentivirus injected brains but drug treatment enhances autophagicclearance via deposition of Aβ₁₋₄₂ in AV-20 and lysosome. N=5 animalsper treatment.

FIG. 21 shows that P-Tau at serine 396 accumulates in AV-10 inlentivirus injected brains but drug treatment enhances autophagicclearance via deposition of p-Tau in AV-20 and lysosome, where it isdegraded. Histograms show p-Tau in subcellular fractions, includingautophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20(autophagosomes) and lysosomes. Wild type or parkin−/− mice wereinjected with lentiviral Aβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kgNilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional)consecutive weeks. Brain tissues were fractionated to isolate AVs andmouse specific. ELISA was performed to determine protein contents.Autophagic clearance is parkin-dependent. N=5 animals per treatment.

FIG. 22 shows that α-synuclein accumulates in AV-10 in lentivirusinjected brains but drug treatment enhances autophagic clearance viadeposition of α-synuclein in AV-20 and lysosome, which containsdegradative enzymes. Histograms show α-synuclein in subcellularfractions, including autophagic vacuole-10 (AV-10;phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes. Wildtype or parkin−/− mice were injected SN with lentiviral a-synuclein for3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinib orDMSO once a day for 3 (additional) consecutive weeks. SN tissues werefractionated to isolate AVs and human specific ELISA was performed todetermine protein contents. Autophagic clearance is parkin-dependent.N=5 animals per treatment.

FIG. 23 shows that P-Tau accumulates in AV-10 in lentivirus injectedbrains but drug treatment enhances autophagic clearance via p-Taudeposition in AV-20 and lysosome, which contains degradative enzymes.Histograms show p-Tau at serine 396 in subcellular fractions, includingautophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20(autophagosomes) and lysosomes. Wild type or parkin−/− mice wereinjected SN with lentiviral α-synuclein for 3 weeks and treated IP with10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3(additional) consecutive weeks. SN tissues were fractionated to isolateAVs and mouse specific ELISA was performed to determine proteincontents. Autophagic clearance is parkin-dependent. N=5 animals pertreatment.

FIG. 24 shows that α-synuclein accumulates in AV-10 in A53T brains butdrug treatment enhances autophagic clearance via α-synuclein depositionin AV-20 and lysosome. Histograms show α-synuclein in subcellularfractions, including autophagic vacuole-10 (AV-10;phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes,containing digestive enzymes. Transgenic A53T mice were injected IP with10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3consecutive weeks. Brain tissues were fractionated to isolate AVs andhuman specific ELISA was performed to determine protein contents. N=5animals per treatment.

FIG. 25 shows that P-Tau accumulates in AV-10 in A53T brains but drugtreatment enhances autophagic clearance via p-Tau deposition in AV-20and lysosome. Histograms show p-Tau at Serine 396 in subcellularfractions, including autophagic vacuole-10 (AV-10;phagophores+autophagosomes), AV-20 (autophagosomes) and lysosomes,containing digestive enzymes. Transgenic A53T mice were injected IP with10 mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once a day for 3consecutive weeks. Brain tissues were fractionated to isolate AVs andmouse specific ELISA was performed to determine protein contents. N=5animals per treatment.

FIG. 26 shows that parkin accumulates in AV-10 in A53T brains but drugtreatment enhances autophagic clearance via parkin deposition in AV-20and lysosome. Histograms show parkin in subcellular fractions, includingautophagic vacuole-10 (AV-10; phagophores+autophagosomes), AV-20(autophagosomes) and lysosomes, containing digestive enzymes. TransgenicA53T mice were injected IP with 10 mg/kg Nilotinib or 5 mg/Kg Bosutinibor DMSO once a day for 3 consecutive weeks. Brain tissues werefractionated to isolate AVs and mouse specific ELISA was performed todetermine protein contents. N=5 animals per treatment.

FIG. 27 is a diagram illustrating how Tyrosine kinase inhibitionincreases parkin activity and facilitates autophagic clearance of p-Tau.This process requires Tau stabilization of intact microtubules. Tyrosinekinase activation, p-Tau accumulation and impaired autophagy arerecognized in neurodegeneration. Decreased parkin solubility andaccumulation with intracellular Aβ and p-Tau in autophagic vacuoles inAD brains occurs, while exogenous parkin facilitates autophagicclearance in animal models.

FIG. 28 shows A) phosphorylated c-Abl at tyrosine 412 (T412) and B)endogenous parkin expression merged in C) hippocampus of 6 month oldC57BL/6 mice treated IP with DMSO daily for 3 weeks. FIG. 28 also showsD) decreased phosphorylated c-Abl at tyrosine 412 (T412) and E)increased endogenous parkin expression merged in F) hippocampus of 6month old C57BL/6 mice treated IP with 5 mg/kg Bosutinib daily for 3weeks.

FIG. 29 shows A) parkin and B) Aβ expression merged in C) cortex of 6month old Tg-APP mice treated with DMSO or 5 mg/kg Bosutinib (D-F) oncea day for 3 weeks. Using a different combination of antibodies (seefigure G-I showing expression of parkin (G) and Aβ (H) in thehippocampus of Tg-APP mice treated DMSO. J-H show the increase in parkinlevel in animals treated for 3 weeks once a day with Bosutinib (J) alongwith decreased plaque levels (K and L) in the hippocampus.

FIG. 30 shows plaque Aβ stained with 6E10 antibody and counterstainedwith DAB in the brain of Tg-APP animals treated IP with DMSO once a dayfor 3 weeks.

FIG. 31 shows plaque Aβ stained with 6E10 antibody and counterstainedwith DAB in the brain of Tg-APP animals treated IP with 5 mg/kgBosutinib once a day for 3 weeks.

FIG. 32 shows that Bosutinib decreases α-synuclein levels in transgenicmice expressing A53T throughout the brain. A-D show human α-synucleinexpression in lentiviral LacZ injected (for 3 weeks) substantia nigrawith A) DMSO and B) 5 mg/kg Bosutinib once a day for 3 weeks. C and Dshow human α-synuclein expression in lentiviral α-synuclein injected(for 3 weeks) substantia nigra with C) DMSO and D) or Bosutinib once aday for 3 weeks. E-H show Tyrosine Hydroxylase (TH) expression inlentiviral LacZ injected (for 3 weeks) substantia nigra with E) DMSO andF) 5 mg/kg Bosutinib once a day for 3 weeks. G and H show TH expressionin lentiviral α-synuclein injected (for 3 weeks) substantia nigra withG) DMSO and H) or Bosutinib once a day for 3 weeks. α-synucleindecreases TH neurons and Bosutinib rescues these cells. I-L show humanα-synuclein expression in A53T mice in I) Cortex, J) Striatum, K)Brainstem and L) Hippocampus treated with DMSO for 3 weeks. M-P showhuman α-synuclein expression in A53T mice in M) cortex, N) striatum, O)brainstem and P) hippocampus treated with 5 mg/kg Bosutinib for 3 weeks.

FIG. 33 provides graphs representing performance on a Morris water mazetest (in seconds) showing that IP treatment with 5 mg/kg Bosutinib oncedaily for 3 weeks improved cognitive behavior in mice injectedbilaterally with lentiviral Aβ₁₋₄₂ for 3 weeks prior to drug treatment.Bosutinib treated mice found the platform (A) but DMSO treated micespent more time in NW area, where they were initially placed or the NEor SW without effectively finding the platform area. Bosutninb improvedcognitive performance in a parkin-dependent manner as the parkin−/− micedid seemed not to learn much. B) shows that Bosutinib treated micetraveled less distance with less speed, but entered the platform areamore than DMSO treated mice.

FIG. 34 shows that tyrosine kinase inhibitors increase parkin activitylevels. A) shows ELISA levels of parkin activity in human M17neuroblastoma cells treated with either 10 mg/kg Nilotinib or 5 mg/kgBosutinib for 24 hrs. N=12. P<0.05. ANOVA, with Neuman Keuls multiplecomparison. An asterisk indicates a significant difference as comparedto DMSO. Bars are mean±SD. B) shows parkin levels (ELISA) in brainlysates of wild type mice injected with lentiviral α-synuclein for 3weeks and then treated with 10 mg/kg Nilotinib once every two days for 3weeks. N=10 animals. P<0.05. ANOVA, with Neuman Keuls multiplecomparison. An asterisk indicates a significant difference as comparedto DMSO. Bars are mean±SD.

FIG. 35 is a Western blot analysis of brain lysates from Tg-APP micetreated with 5 mg/kg Bosutinib for 3 additional weeks. These blots showdecreased levels of c-Abl, increased parkin and alteration of differentmolecular markers of autophagy, indicating that Aβ alters normalautophagy and Bosutinib boosts autophagy to clear Aβ₁₋₄₂.

FIG. 36 is a Western blot analysis of brain lysates from Tg-APP micetreated with 5 mg/kg Bosutinib for 3 weeks. These blots show alterationsin the levels of molecular markers of autophagy.

FIG. 37 is a Western blot analysis of brain lysates from Tg-APP micetreated with 5 mg/kg Bosutinib for 3 additional weeks. These blots showdecreased levels of C-terminal fragments (CTFs) and phospho-tyrosine.

FIG. 38 is a Western blot analysis of brain lysates from Tg-APP micetreated with 5 mg/kg Bosutinib once a day for additional weeks. Theseblots show decreased levels of different Tau isotopes.

FIG. 39 is a Western blot analysis of brain lysates from wild type miceexpressing lentiviral Aβ₁₋₄₂ (3 weeks) with and without Bosutinib (5mg/kg) treatment for 3 additional weeks. These blots show levels ofdifferent molecular markers of autophagy, indicating that Aβ₁₋₄₂ altersnormal autophagy and Bosutinib boosts autophagy to clear Aβ₁₋₄₂.

FIG. 40 is a Western blot analysis of brain lysates from wild type miceexpressing lentiviral Aβ₁₋₄₂ (3 weeks) with and without Bosutinibtreatment for 3 additional weeks. These blots show decreased levels ofubiquitin (top blot) and pan phospho-tyrosine (second blot) and SIAH2,suggesting that Bosutinib is a broad tyrosine kinase inhibitor.

FIG. 41 is a Western blot analysis of brain lysates from wild type miceexpressing lentiviral Aβ₁₋₄₂ (3 weeks) with and without Bosutinibtreatment for 3 additional weeks. These blots show decreased levels ofdifferent Tau isotopes.

FIG. 42 is a Western blot analysis of brain lysates from wild type miceexpressing lentiviral α-synuclein (3 weeks) with and without Bosutinibtreatment for 3 additional weeks. Blots show in order increasedα-synuclein in lentiviral synuclein injected animals, along withdecreased c-Abl levels and phosphorylation, increased parkin levels andmarkers of autophagy, including P62, HDAC6, LC3 and ATG12 compared toloading controls tubulin and MAP2.

FIG. 43 shows that parkin is insoluble in post-mortem striatum of humanPD patients. A) Histograms represent ELISA measurement of human parkinin the caudate of PD patients and control subjects. B) is a WB analysison 4-12% SDS-NuPAGE gel of soluble human post-mortem striatal lysates inPD patients and control subjects, showing parkin (1st blot) andubiquitinated proteins (2nd blot) compared to actin loading control. C)Histograms represent quantification of blots. D) is a WB analysis on4-12% SDS NuPAGE gel showing the levels of insoluble parkin (1st blot),phospho-parkin (2nd blot), ubiquitinated proteins (3rd blot), and actin(4th blot). E) Histograms represent quantification of blots. Asterisksindicate a significant difference. F) Box plot represents individualsamples of human PD patients and age-matched controls. Histograms aremean±SD expressed as % to control. ANOVA, Neumann Keuls with multiplecomparison, or non-parametric t-Test. P<0.05. N=12 PD patients and 7control subjects.

FIG. 44 shows immunostaining of human tissues with human and GFAβantibodies. Immunostaining of 20 μm thick paraffin embedded seriallysectioned brains with A) human anti-parkin (PRK8) staining andcounterstaining with nuclear marker DAPI showing cytosolic protein, B)co-staining with parkin and glial marker GFAβ showing parkin expressionin astrocytes, C) TH staining in the caudate of a control subject, D)parkin staining and counterstaining with nuclear marker DAPI showingcytosolic protein, E). co-staining with parkin and glial marker GFAβshowing parkin expression in astrocytes, F). TH staining in the caudateof a PD/AD patient, G) parkin staining and counterstaining with DAPIshowing cytosolic protein, H) co-staining with parkin and glial markerGFAβ showing parkin expression in astrocytes, I) TH staining in themidbrain/SN of a control subject, J) parkin staining and counterstainingwith DAPI showing cytosolic protein, K) co-staining with parkin andglial marker GFAβ showing parkin expression in astrocytes, L) THstaining in the midbrain/SN of a PD patient. M). human anti-parkin(AB5112) staining and counterstaining with nuclear marker DAPI showingcytosolic protein, N) co-staining with parkin and glial marker GFAβshowing parkin expression in astrocytes, O) TH staining in the caudateof a control subject.

FIG. 45 shows subcellular fractionation in frozen human PD braintissues. A) shows human anti-parkin (AB5112) staining andcounterstaining with nuclear marker DAPI showing cytosolic protein. B)shows neuronal marker MAP-2 staining and DAPI and C) shows merged parkinand MAP-2 in stained serial sections. D) shows TH in the midbrain/SN ofa control subject. E) shows human anti-parkin (AB5112) staining andcounterstaining with nuclear marker DAPI showing cytosolic protein. F)shows neuronal marker MAP-2 staining and DAPI and G) shows merged parkinand MAP-2 in serial sections stained with H) TH in the midbrain/SN of aPD with Dementia patient. I) shows a WB analysis on 4-12% SDS NuPAGE gelof human striatal lysates showing expression of LC3-I and LC3-II (firstpanel), LC3-B (second panel) compared to actin loading control (bottompanel) J) shows histograms representing densitometry analysis of blots.K) shows a Western blot in subcellular extracts showing LC3-B in AV-10and AV-20 and LAMP-3 in lysosomal fraction, as well as mitochondrialmarker COX-IV and nuclear marker PARP-1. Graphs represent subcellularfractionation and ELISA measurement of L) human α-synuclein, M) humanparkin and N) human p-Tau (AT8). Asterisks indicate significantlydifferent to control. ANOVA, Neumann Keuls with multiple comparison,P<0.05. N=12 PD patients and 7 control subjects.

FIG. 46 shows lentiviral expression of α-synuclein leads to p-Tau andparkin activity reverses these effects. A) is aWB analysis on 4-12%SDS-NuPAGE gel of rat striatal extracts showing levels of parkin (topblot) and α-synuclein (middle blot) expression and actin levels (lowerblot). B) shows histograms representing quantification of humanα-synuclein levels by ELISA. C) shows histograms representingquantification of human parkin activity. D) is an ELISA measurement ofrat p-Tau. Thioflavin-S staining of 20 μm striatal sections inlentiviral E) parkin, F) α-synuclein and G) parkin+α-synuclein injectedbrains. Human α-synuclein staining of 20 μm sections cut serially withthe thioflavin-S sections is shown in for lentiviral K) parkin, L)α-synuclein and M) parkin+α-synuclein injected brains. Asterisksindicate significantly different. Histograms are mean±SD expressed as %control. ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=8animals per treatment for WB and ELISA, 8 for IHC.

FIG. 47 shows that wild type, but not T240R, parkin reversesα-synuclein-induced accumulation of autophagosomes. Electron micrographsof striatal sections in rat brains injected with A) Lentiviral LacZ(Lv-LacZ) as control, B) Lentiviral α-synuclein (Lv-Syn), C) Lentiviralparkin+lentiviral-α-synuclein (Lv-Syn+Lv-Par), vacuoles contain debrisand D) Lentiviral α-synuclein+lentiviral T240R (Lv-Syn+Lv-T240R).Asterisk indicates autophagic vacuoles. N=8. Graphs representsubcellular fractionation and ELISA measurement of E) α-synuclein and F)p-Tau in gene transfer animal models. ANOVA, Neumann Keuls with multiplecomparison, P<0.05. N=5 animals per treatment for subcellularfractionation.

FIG. 48 shows that functional parkin, not mutant T240R reversesα-synuclein alteration of normal autophagy. A) shows a WB analysis on4-12% SDS NuPAGE gel of rat striatal lysates showing expression ofbeclin (first panel), Atg7 (second panel) and Atg12 (third panel)compared to actin loading control (bottom panel) in animals injectedwith Lv-LacZ, Lv-Par, Lv-Syn and Lv-Par+Lv-Syn. B) shows aWB analysis ofrat striatal brain lysates showing expression of LC3-B (first panel),and HDAC6 (second panel) compared to actin loading control (bottompanel) in animals injected with Lv-LacZ, Lv-Par, Lv-Syn andLv-Par+Lv-Syn. Staining of 20 μm thick cortical brain sections injectedwith C) Lentiviral parkin (Lv-Par), D) Lentiviral α-synuclein (Lv-Syn)E) Lentiviral parkin+lentiviral α-synuclein (Lv-Par+Lv-Syn) and F)Lentiviral T240R+lentiviral α-synuclein (Lv-T240R+Lv-Syn) is shown. G)shows histograms representing stereological counting of LC3-B positivecells in the striatum. H) is a Western blot analysis on 4-12% SDS NuPAGEgel with P62 antibody. Asterisks indicate a significant difference.Histograms are mean±SD converted to % control. ANOVA, Neumann Keuls withmultiple comparison, P<0.05. N=8 animals per treatment for WB and ELISA,8 for IHC.

FIG. 49 shows that parkin is increased in AD brains. A) shows aWBanalysis on 4-12% SDS-NuPAGE gel of human post-mortem cortical lysatesin AD. B) shows histograms representing human parkin levels measured byELISA. C) is aWB analysis on 4-12% SDS-NuPAGE gel showing expressionlevel of parkin's possible targets for degradation, includingubiquitinated proteins (top blot), tubulin (2nd blot) and Cyclin E (3rdblot) and actin (4th blot). D) shows histograms representing blotquantification by densitometry. E) is aWB analysis on 4-12% SDS-NuPAGEgel showing insoluble proteins extracted in 4M urea, including totalparkin (top blot) and phosphorylated parkin at Serine 378 (2nd blot) andactin (3rd blot). F) shows Histograms representing blot quantificationby densitometry. Asterisks indicate a significant difference. Histogramsare mean±SD expressed as % control. All bands were quantified relativeto actin levels. ANOVA, Neumann Keuls with multiple comparison, P<0.05.

FIG. 50 shows increased intraneuronal Aβ₁₋₄₂ and parkin co-localizationin the hippocampus of AD brains. IHC of paraffin embedded 30 μm thicksections of human hippocampus from control subject (case #1252) stainedwith A) Human anti-Aβ₁₋₄₂ antibody+DAPI and B) Anti-parkin antibody+DAPIare shown. C) is a merged figure showing co-staining of Aβ₁₋₄₂ andparkin. IHC of sections of hippocampus from AD patient (case #1774)stained with D) Human anti-Aβ₁₋₄₂ antibody+DAPI and E) Human anti-parkinantibody+DAPI are shown. F) is a merged figure showing co-staining ofAβ₁₋₄₂ and parkin. IHC of sections of hippocampus from AD patient (case#1861) stained with G) 4G8 anti-Aβ₁₋₄₂ antibody+DAPI and H) humananti-parkin antibody+DAPI are shown. and I) is a merged figure showingco-staining of (4G8) Aβ₁₋₄₂ and parkin.

FIG. 51 shows that parkin co-localizes with intraneuronal Aβ₁₋₄₂ in thecortex of AD brains. IHC of paraffin embedded 30 μm thick sections ofhuman entorhinal cortex from AD patient (case #1833) stained with A)human anti-Aβ₁₋₄₂ antibody+DAPI and B) anti-parkin antibody+DAPI areshown. C) is a merged figure showing co-staining of Aβ₁₋₄₂ and parkin.IHC of sections of human neocortex from AD patient (case #1851) stainedwith D) human anti-Aβ₁₋₄₂ antibody+DAPI and E) anti-parkin antibody+DAPIare shown. F) is a merged figure showing co-staining of Aβ₁₋₄₂ andparkin. IHC of sections of necortex from AD patient (case #1861) stainedwith G) 4G8 anti-Aβ₁₋₄₂ antibody+DAPI and H) human anti-parkinantibody+DAPI are shown. I) is a merged figure showing co-staining of(4G8) Aβ₁₋₄₂ and parkin.

FIG. 52 shows that parkin, Aβ₁₋₄₂ and p-Tau accumulate in autophagicvacuoles of AD brains. A) is aWB analysis on 4-12% SDS-NuPAGE gel ofhuman post-mortem cortical lysates in AD probed with anti-LC3 antibodyshowing LC3-I and LC3-II (1st blot) and LC3-B (2nd blot) and actin (3rdblot). B) shows histograms representing blot quantification bydensitometry. C) is aWB analysis of Metrazimide-isolated fractions fromfrozen brain tissue showing lysosomal marker LAMP-3 in the floatingfraction and detection of LC3-B in AV-10 and AV-20. Graphs representELISA measurement in autophagic vacuoles of human D) Aβ₁₋₄₂, E) Aβ₁₋₄₀,F) p-Tau (AT8) and G) parkin. Asterisks indicate a significantdifference. Histograms are mean±SD expressed as % control. All bandswere quantified relative to actin levels. ANOVA, Neumann Keuls withmultiple comparison, P<0.05.

FIG. 53 shows that parkin decreases the level of lentiviral Aβ₁₋₄₂ andp-Tau in gene transfer animal models. A) is aWB analysis on 4-12% SDSNuPAGE gel showing the expression levels of parkin and Aβ₁₋₄₂, analyzedwith a synthetic peptide as a molecular weight and antibody control. B)shows histograms represent quantification of human parkin by ELISA. C)shows a human Aβ₁₋₄₂ ELISA 2 weeks after lentiviral injection. D) showsELISA measurement of rat p-Tau 2 and 4 weeks post-injection.Thioflavin-S staining of 20 μm cortical sections in lentiviral E) LacZ,F) Aβ₁₋₄₂ and G). parkin+ Aβ₁₋₄₂ injected brains is also shown.Asterisks indicate a significant difference. Histograms are mean±SDexpressed as % to control. All bands were quantified relative to actinlevels. ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=8animals per treatment for WB and ELISA, 8 for IHC.

FIG. 54 shows that parkin clears Aβ₁₋₄₂-induced accumulation ofautophagic vacuoles. Electron micrographs of cortical sections in ratbrains injected with A) Lentiviral LacZ (Lv-LacZ) as control, B)lentiviral parkin (Lv-Par), C) lentiviral A Aβ₁₋₄₂ (Lv-A Aβ₁₋₄₂) (arrowsindicate vacuoles) and D) lentiviral parkin+lentiviral A Aβ₁₋₄₂ (Lv-AAβ₁₋₄₂+Lv-Par) (vacuole contains debris) are shown. N=8. Graphsrepresent subcellular fractionation (Blot) and ELISA measurement of E)Aβ₁₋₄₂ and F) p-Tau in gene transfer animal models. All bands werequantified relative to actin levels. ANOVA, Neumann Keuls with multiplecomparison, P<0.05. N=5 animals per treatment for subcellularfractionation.

FIG. 55 shows that intracellular Aβ₁₋₄₂ impairs normal autophagy andparkin facilitates autophagic clearance. A) is aWB analysis on 4-12% SDSNuPAGE gel of rat cortical lysates showing expression of beclin (firstpanel), Atg7 (second panel) and Atg12 (third panel) and actin loadingcontrol (bottom panel) in animals injected with Lv-LacZ, Lv-Par,Lv-Aβ₁₋₄₂ and Lv-Par+Lv-Aβ₁₋₄₂. B) is aWB analysis of rat cortical brainlysates showing expression of LC3-B (first panel), and HDAC6 (secondpanel) and actin loading control (bottom panel) in animals injected withLv-LacZ, Lv-Par, Lv-Aβ₁₋₄₂ and Lv-Par+Lv-Aβ₁₋₄₂. Staining of 20 μm thickcortical brain sections injected with C) lentiviral LacZ (Lv-LacZ), D)lentiviral parkin (Lv-Par) E) lentiviral Aβ₁₋₄₂ (Lv-Aβ₁₋₄₂) and F)lentiviral parkin+lentiviral Aβ₁₋₄₂ (Lv-Par+Lv-Aβ₁₋₄₂) are shown. G)shows histograms representing stereological counting of LC3-B positivecells in the cortex. H) is a WB analysis of 4-12% SDS NuPAGE gel showingP62 levels. Asterisks indicate a significant difference. Histograms aremean±SD expressed as % control. All bands were quantified relative toactin levels. ANOVA, Neumann Keuls with multiple comparison, P<0.05. N=8animals per treatment for WB and ELISA, 8 for IHC.

FIG. 56 shows that c-Abl activation is associated with accumulation ofα-synuclein. A WB on 10% SDS-NuPAGE gel shows A) lentiviral α-synucleinexpression (1st blot), total c-Abl (2nd blot) and tyrosine 412 (T412)phosphorylated c-Abl (3rd blot) and actin (N=9). B) shows total c-Abl(1st blot) T412 c-Abl (2nd blot) and actin in human post-mortem striatalextracts, N=9 PD and 7 controls, p<0.02, two-tailed t-test. C) showsdensitometry of human WBs. D) is aWB on 4-12% SDS-NuPAGE gel that showstotal c-Abl (1st blot) and tyrosine 412 (T412) phosphorylated c-Abl (2ndblot), and mouse α-synuclein expression (3rd blot) and actin (N=9). E)is a graph representing quantification of Mass Spec analysis of brainNilotinib (N=5/time point). Graphs represent caspase-3 activity in F)lentiviral α-synuclein and LacZ injected mice (N=14) with and withoutNilotinib, and G) 6-8 month old transgenic A53T mice (N=15) and wildtype age-matched controls (N=64) with and without Nilotinib.*Significantly different, ANOVA, Neumann Keuls multiple comparison,p<0.05.

FIG. 57 shows that Nilotinib clears α-synuclein and protects SN Tyrosinehydroxylase (TH) neurons. Immunohistochemical staining of 20 μm thickbrain sections show human α-synuclein in A) lentiviral injectedLacZ+Nilotinib mice, B) mice injected with lentiviral α-synuclein intothe SN and treated with DMSO and C) mice injected with lentivirala-synuclein and treated with Nilotinib. Immunohistochemical staining of20 μm thick brain sections show Tyrosine Hydroxylase in D) lentiviralinjected LacZ+Nilotinib mice, G is higher magnification from a differentanimal and E) mice injected with lentiviral α-synuclein and treated withDMSO. H) is higher magnification from a different animal. F) shows miceinjected with lentiviral α-synuclein and treated with Nilotinib. I) ishigher magnification from a different animal. J) shows Nisslcounter-stained cells in LacZ+Nilotinib, K). α-synuclein+DMSO and L).α-synuclein+Nilotinib.

FIG. 58 shows that Nilotinib clears accumulation of autophagic vacuolesin SN of lentiviral α-synuclein mice. A) Transmission electronmicroscopy of SN neurons shows accumulation of cytosolic debris andautophagic vacuoles (AVs) in Lentiviral α-synuclein expressing mice withDMSO treatment, B) shows appearance of larger AVs in Nilotinib treatedmice, C) shows accumulation of cytosolic debris and autophagic vacuoles(AVs) in Lentiviral α-synuclein expressing mice with DMSO treatment, D)shows appearance of larger AVs in Nilotinib treated mice, E) showsaccumulation of cytosolic debris and autophagic vacuoles (AVs) inLentiviral α-synuclein expressing mice with DMSO treatment, F) showsappearance of larger AVs in Nilotinib treated mice.

FIG. 59 shows that Nilotinib attenuates α-synuclein levels in A53T mice.Immunohistochemical staining of 20 μm thick brain sections showsabundant expression of human α-synuclein in 6-8 month old transgenicA53T mice treated with DMSO in the A) striatum, B) brainstem C) cortexand D) hippocampus of different animals. Daily IP injection of Nilotinibfor 3 weeks shows decrease of human α-synuclein in the E) striatum, F)brainstem G) cortex and H) hippocampus.

FIG. 60 shows that Nilotinib activates parkin and induces autophagicclearance. A) is a graph representing MTT-based cell viability in humanM17 neuroblastoma cells (N=12) transfected with Aβ₁₋₄₂ (or LacZ) cDNAfor 24 hr, and then treated with 10 μM Nilotinib for an additional 24hr. B) is a graph representing proteasome activity via Chymotrypsin-likeassays using 20 μM 20S proteasome inhibitor lactacystin as a specificitycontrol in human neuroblastoma cells (N=12) with and without Nilotinib.C) is a Human Aβ₁₋₄₂ ELISA before and after Nilotinib treatment in B35rat neuroblastoma cells (N=12) in media, soluble (STEN buffer) andinsoluble (30% formic acid) lysates in the presence and absence of shRNAbeclin-1. D) is aWB of soluble cell lysates (from C) showing beclin-1,parkin and LC3 levels with and without Nilotinib (N=12). E) is a graphrepresents parkin E3 ubiquitin ligase function in B35 neuroblastomacells treated with DMSO or Nilotinib for 24 hr. Recombinant E1-E2-E3(positive) or K0 (negative) were used as specificity controls. F) is agraph representing caspase-3 activity in 1 year old C57BL/6 (N=64) (wildtype) or parkin−/− mice (N=16-19) injected with lentiviral Aβ₁₋₄₂ andtreated (IP) with 10 mg/kg for 3 weeks. *Significantly different, ANOVAwith Neumann Keuls multiple comparison, p<0.05.

FIG. 61 shows that Nilotinib clearance of brain amyloid is associatedwith parkin activation. A graph represents ELISA levels of A) solubleand insoluble human Aβ₁₋₄₂ and B) ELISA levels of soluble and insolublehuman Aβ1-40 in the brain of 8-12 months old Tg-APP mice (N=9) injected(IP) with 10 mg/kg once a day for 3 weeks. C) is a graph that representsELISA levels of mouse p-Tau in the brain of 8-12 months old Tg-APP mice(N=9). D) is a graph tat represents ELISA levels of soluble andinsoluble mouse parkin in the brain of 8-12 months old Tg-APP mice (N=9)injected (IP) with 10 mg/kg (daily for 3 weeks) and parkin−/− brainextracts as specificity control. E) is aWB analysis on 4-12% SDS Nu-PAGEgels of brain extracts from Tg-APP treated with Nilotinib or DMSOshowing APP, c-Abl, p-c-AB1 and CTFs and MAP-2 as control (N=11). F) isaWB of post-mortem cortical extracts of AD patients (N=12 AD and 7control) on 10% SDS Nu-PAGE and G) is a graph that representsdensitometry and ratio of c-Abl and p-c-Abl and parkin. * Significantlydifferent, non-parametric t-test, P<0.05. Also shown is a graphrepresenting ELISA levels of H) soluble and insoluble human Aβ₁₋₄₂, andI) ELISA levels of mouse p-Tau in the brain of mice (N=9) injected (IP)with 10 mg/kg (3 weeks).

* Significantly different, ANOVA with Neumann Keuls multiple comparison,p<0.05.

FIG. 62 shows that Nilotinib promotes autophagic clearance of amyloid.WB of brain extracts on 4-12% Nu-Page SDS gels are shown for A) inlentiviral Aβ₁₋₄₂ in wild type mice±Nilotinib showing, c-Abl, p-c-Abl,LC3-B and LC3 relative to MAP-2 and B) parkin, beclin-1, Atg-5 and 12relative to tubulin (N=9). Western blot analysis of brain extracts on10% Nu-Page SDS gels for C) Tg-APP±Nilotinib showing, parkin, LC3B, LC3,Atg-5 and beclin-1 relative to tubulin and D) total Tau, ATB, AT180, Ser396 and Ser 262 relative to actin (N=12) are also provided. E) is aWestern blot analysis of brain extracts on 4-12% Nu-Page SDS gels inlentiviral Aβ₁₋₄₂ in parkin−/− mice±Nilotinib showing, parkin, beclin-1,LC3 and LC3A relative to tubulin and F) is a Western blot analysis ofAtg-5 and Atg12 relative to MAP-2 (N=7).).*Significantly different,ANOVA with Neumann Keuls multiple comparison, p<0.05.

FIG. 63 shows that Nilotinib increases parkin level and decreases plaqueload. Staining of 20 μm brain sections shows plaque formation withinvarious brain regions in A-D) Tg-APP+DMSO and E-H) Nilotinib group aftera 3-week treatment. Staining of 20 μm thick brain sections shows I)parkin and J) Aβ₁₋₄₂ K) is a merged figure in hippocampus of Tg-APP miceafter 3 weeks of DMSO treatment. L) shows parkin, M) shows Aβ₁₋₄₂ and N)is a merged figure in hippocampus of Tg-APP mice after 3 weeks ofNilotinib treatment. 0) shows parkin, P) shows Aβ₁₋₄₂ and Q) is amergedfigure in the cortex of Tg-APP mice after 3 weeks of DMSO treatment. R)shows parkin, S) shows Aβ₁₋₄₂ and T) is amerged figure in cortex ofTg-APP mice after 3 weeks of Nilotinib treatment. Staining of 20 μmbrain sections shows intracellular Aβ₁₋₄₂ within the U). hippocampus oflentiviral Aβ₁₋₄₂ injected mice, inset higher magnification, and V)Nilotinib clearance of intracellular Aβ₁₋₄₂ (inset is highermagnification). Staining of 20 μm brain sections shows intracellularAβ₁₋₄₂ within the W) cortex of lentiviral Aβ₁₋₄₂ injected mice, insethigher magnification, and X) Nilotinib clearance of intracellular Aβ₁₋₄₂(inset is higher magnification).

FIG. 64 shows that Nilotinib eliminates plaques in lentiviral Aβ₁₋₄₂injected wild type but not parkin−/− mice. Staining of 20 μm brainsections shows plaque formation within various brain regions indifferent A-C) lentiviral Aβ₁₋₄₂+DMSO wild type mice and D-F) Nilotinibgroup after 3-week treatment. G-I) show lentiviral Aβ₁₋₄₂+DMSO inparkin−/− mice and J-L) show the Nilotinib group after 3-week treatment.Transmission electron microscopy shows autophagic defects in differentlentiviral Aβ₁₋₄₂+DMSO wild type brains within M) hippocampus showingdistrophic neurons, N) cortex showing accumulation of autophagicvacuoles, O) hippocampus showing enlarged lysosomes. LentiviralAβ₁₋₄₂+Nilotinib wild type brains within P) hippocampus, Q) cortexshowing clearance of autophagic vacuoles, R) hippocampus. LentiviralAβ₁₋₄₂±Nilotinib in parkin−/− brains within S&V) hippocampus showingdistrophic neurons, T&W) cortex showing accumulation of autophagicvacuoles and U&X), hippocampus showing accumulation of autophagicvacuoles, are also shown.

FIGS. 65A-E show that Nilotinib ameliorates cognition in aparkin-dependent manner. FIG. 65A) represents the results of a Morriswater maze test after 4 days of training (trials) in lentiviralAβ₁₋₄₂-injected±Nilotinib wild type (N=14) and parkin−/− (N=7) mice.FIG. 65B) shows graphs representing the total number of entry intoplatform area and distance travelled. FIG. 65C) represents the resultsof a Morris water maze test after 4 days of training (trials) inTg-APP±Nilotinib (N=12) mice, including heat maps for each group showingoverall performance. FIG. 65D) shows graphs representing total number ofentry into platform area and distance travelled. FIG. 65E) representsthe results of an object recognition test in Tg-APP±Nilotinib (N=12) andlentiviral Aβ₁₋₄₂-injected±Nilotinib in parkin−/− (N=7). The recognitionindex was calculated as (time exploring one of the objects/timeexploring both objects)×100 for acquisition session, and (time exploringnew object/time exploring both familiar and novel objects)×100 for therecognition session given 1.5 hrs later.

*Significantly different, ANOVA with Neumann Keuls multiple comparison,P<0.05, Significant effect of Nilotinib on recognition in Tg-APP group,pairwise T-test p<0.001.

FIG. 66 shows that Nilotinib increases parkin level and crosses theblood brain barrier. Parkin levels by ELISA in wild type mice andlentiviral Aβ₁₋₄₂±Nilotinib using parkin−/− brain extracts as aspecificity control (N=12) are shown.

FIG. 67 shows that Nilotinib eliminates thioflavin-S staining.Thioflavin staining of 20 μm brain sections shows plaque formationwithin various brain regions in different A-D) Tg-APP+DMSO and E-H)Nilotinib group after 3-week treatment.

FIG. 68 shows that parkin ubiquitinates Aβ₁₋₄₂ to mediate itsdegradation. Staining of 20 μm thick sections shows formation of6E10-positive plaques in Aβ₁₋₄₂ expressing group 6 weeks post-injectionin A) Aβ₁₋₄₂ wild type mice+DMSO, B) Aβ₁₋₄₂ wild type mice+Nilotinib, C)Aβ₁₋₄₂ parkin−/− mice+DMSO and D) Aβ₁₋₄₂ parkin−/− mice+Nilotinib.Higher magnification showing 6E10 positive cells are provided in E)Aβ₁₋₄₂ wild type mice+DMSO, F) Aβ₁₋₄₂ wild type mice+Nilotinib, G)Aβ₁₋₄₂ parkin−/− mice+DMSO, H) Aβ₁₋₄₂ parkin−/− mice+Nilotinib. I) showsa graph representing quantification of plaque size using image J todelineate boundaries around individual plaques using 15-25 plaques (2plaques per animal) and J) shows stereological counting of Aβ₁₋₄₂positive cells (N=12 animals). K) is a graph representing parkinactivity (N=6). * Significantly different, ANOVA with Neumann Keulsmultiple comparison, p<0.05.

FIG. 69 shows that TDP-43 inhibits proteasome activity and alters parkinlevels. Western blot analysis of soluble cortical brain lysates fromdifferent litters of mixed male and female TDP-43 transgenic mice andnon-transgenic control littermates on 4-12% SDS NuPAGE gel are providedshowing A) human TDP-43 levels probed with 2E2-D3 antibody (1st blot),total parkin (2nd blot), ubiquitin (3rd blot) and actin (4th blot)levels. B) shows that the pellet was re-suspended in 4M urea to extractthe insoluble protein fraction and Western blot was performed showinginsoluble parkin (1st blot) and insoluble TDP-43 (2nd blot) compared toactin loading control (3rd blot). C) shows a densitometry analysis of Aand B blots showing soluble and insoluble parkin protein levelsnormalized to actin and the ratio of soluble to insoluble parkin. D)shows an ELISA measurement of parkin level in soluble (STEN extracts)and insoluble (4M Urea) brain extracts compared to parkin−/− brainextracts as a specificity control. E) is a Western blot analysis ofcortical brain lysates on 4-12% SDS NuPAGE gel showing soluble proteinlevels of the E3 ubiquitin ligase SIAH2 (1st blot) and its targetprotein HIF-1α (2nd blot) compared to actin loading control. F) showsdensitometry analysis of blots in D normalized to actin control, N=4,ANOVA with Neumann Keuls, P<0.05. G) shows Western blot analysis of M17cell lysates on 4-12% SDS NuPAGE gel showing human TDP-43 levels (1stblot), total parkin (2nd blot), ubiquitin (3rd blot) SIAH2 (4th blot)and actin levels (5th blot) in cells expressing TDP-43 and wild typeparkin. H) is a Western blot analysis of M17 cell lysates on 4-12% SDSNuPAGE gel showing human TDP-43 levels (1st blot), total parkin (2ndblot), ubiquitin (3rd blot) SIAH2 (4th blot) and actin levels (5th blot)in cells expressing LacZ and wild type parkin. I) shows Histogramsrepresent the chymotrypsin proteasome activity in M17 neuroblastomacells. *Significantly different, ANOVA, Neumann Keuls, P<0.05, N=6 forcells.

FIG. 70 shows that Lentiviral expression of TDP-43 in rat motor cortexresults in detection of TDP-43 in preganglionic cervical spinal cordinter-neurons. Staining of 20 μm thick sections from rat brain injectedwith lentiviral TDP-43 in the right hemisphere and lentiviral LacZ inthe left hemisphere showing A) neurons in rat motor cortex stained withanti-TDP-43 antibody that detects both human and rat TDP-43 andDAPI-stained nuclei in lentiviral LacZ-injected and B) TDP-43 injectedhemisphere are shown. C) shows that neurons in rat motor cortex stainedwith anti-TDP-43 antibody that detects human TDP-43 and DAPI-stainednuclei in lentiviral LacZ-injected and D) TDP-43 injected hemisphere. E)is a schematic representation of injected motor cortex relative tocontralateral spinal cord region and dorso-cortical spinal tract (DCST).Staining of 20 μm thick sections showing preganglionic cervical spinalcord inter-neurons stained with F). hTDP-43 mouse monoclonal antibody(Abnova) that recognizes human TDP-43 and G). Anti-TDP-43 rat polyclonalantibody (ProteinTech) that recognizes both human and rat, andDAPI-stained nuclei contralateral to lentiviral TDP-43-injected cortexand H (TDP-43) and I (hTDP-43) contralateral to LacZ injected hemisphereare shown. Staining of 20 μm thick sections showing fibers in DCSTstained with J) mouse monoclonal hTDP-43 and DAPI and K) rabbitpolyclonal anti-TDP-43 antibody DAPI contralateral to lentiviralTDP-43-injected cortex are also shown. L shows TDP-43 and M showshTDP-43. TDP-43 staining and DAPI in DCST contralateral to LacZ injectedhemisphere was also performed. N) shows toluidine blue stained DCSTcontralateral to lentiviral TDP-43-injected cortex compared to O) LacZinjected hemisphere. Lv: lentivirus.

FIG. 71 shows that lentiviral parkin increases cytosolic co-localizationof ubiquitin and TDP-43. Staining of 20 μm thick sections from rat braininjected with lentiviral TDP-43 in the right hemisphere and lentiviralLacZ in the left hemisphere shows A) neurons in rat motor cortex stainedwith mouse monoclonal (Millipore) anti-parkin, B) rabbit polyclonalanti-TDP-43 antibodies, C) parkin, TDP-43 and DAPI in lentiviralLacZ-injected hemisphere. D) shows neurons in rat motor cortex stainedwith mouse monoclonal anti-ubiquitin and E) rabbit polyclonalanti-TDP-43 antibodies. F) shows ubiquitin, TDP-43 and DAPI inlentiviral TDP-43-injected hemisphere. G) shows neurons in rat motorcortex stained with mouse monoclonal anti-parkin and H) rabbitpolyclonal anti-TDP-43 antibodies. I) shows parkin, TDP-43 and DAPI inanimals co-injected with lentiviral TDP-43 and parkin. J) shows neuronsin rat motor cortex stained with mouse monoclonal anti-ubiquitin and K)rabbit polyclonal anti-TDP-43 antibodies. L) shows ubiquitin, TDP-43 andDAPI stained nuclei in animals co-injected with lentiviral TDP-43 andparkin. Neurons in rat motor cortex stained with M) mouse monoclonalanti-parkin antibodies, N) rabbit polyclonal anti-TDP-43 antibodies, andO) parkin, TDP-43 and DAPI stained nuclei in animals injected withlentiviral parkin alone are shown. Lv: lentiviral.

FIG. 72 shows that parkin mediates K48 and K63-linked ubiquitination ofTDP-43. Western blot of input samples from cortical brain lysatesanalyzed on 4-12% SDS NuPAGE gel show A) parkin expression levels (1stblot), ubiquitin bound protein levels (2nd blot) and TDP-43 levels (3rdblot), compared to actin loading control in rat cortex injected withlentiviral LacZ, TDP-43, parkin, TDP-43+parkin and TDP-43+T240R mutant.A total of 100 mg cortical brain samples were immuno-precipitated usingrabbit polyclonal anti-TDP-43 and probed (1:1000) with anti-ubiquitinantibody (4th blot) compared to actin loading control (5th blot) frominput samples. B) shows a Western blot of input samples andimmuno-precipitated parkin (top blot) and TDP-43 (bottom blot) fromtransgenic mice used to measure parkin E3 ubiquitin ligase activity. C)shows histograms representing parkin E3 ubiquitin ligase activity in thepresence and absence of human TDP-43 immuno-precipitated from TDP-43transgenic mice, compared to E3 ubiquitin ligase activity usingrecombinant parkin (sPar), poly-ubiquitin chain as control and asynthetic E1-E2-E3 control combination. N=8, P<0.05, ANOVA NeumannKeuls. D) is aWB analysis showing ubiquitinated TDP-43 in the presenceof K48 and K63 and E) is aWB analysis showing ubiquitinated parkin atK48 and K63. F) shows histograms representing the chymotrypsinproteasome activity in fresh cortical brain lysates from rats injectedwith lentiviral LacZ, parkin, TDP-43 and TDP-43+parkin. * indicates asignificant difference, ANOVA, Neumann Keuls, P<0.05, N=8. G). Westernblot analysis of cortical brain lysates on 4-12% SDS NuPAGE gel showingHDAC6 (1st blot) and P62 levels (2nd blot) and actin control (3rd blot)are provided. H) is a densitometry analysis of blots in E from genetransfer animal models. * Indicates significantly different, ANOVA,Neumann Keuls, P<0.05, N=8.

FIG. 73 shows that TDP-43 forms a multi-protein complex with parkin andHDAC6. Western blot of input samples from cortical brain lysates intransgenic A315T mice and control littermates analyzed on 4-12% SDSNuPAGE gel showing A) shows human TDP-43 expression levels (1st blot)and immuno-precipitation of TDP-43 showing TDP-43 (2nd blot), parkin(3rd blot) and HDAC6 (4th blot) forming a protein complex. B) representsthe reverse immune-precipitation experiment, where Western blot of inputsamples from cortical brain lysates in transgenic A315T mice and controllittermates analyzed on 4-12% SDS NuPAGE show parkin expression levels(1st blot) and immuno-precipitation of parkin showing TDP-43 (2nd blot),parkin (3rd blot) and HDAC6 (4th blot). GFP fluorescence and nuclearDAPI-staining in living human M17 neuroblastoma cells C) shows cellstransfected with GFP-TDP-43 alone showing GFP fluorescence within thenucleus. D & E) show cells transfected with GFP-TDP-43 and parkinshowing GFP fluorescence in cytosol and cellular processes. Inset in Dshows higher magnification. F) shows cells transfected with GFP-TDP-43and parkin treated with 5 μM HDAC6 inhibitor, tubacin for 24 hoursshowing GFP fluorescence within DAPI-stained nuclei. G). shows cellstransfected with GFP-TDP-43 for 24 hours and treated with tubacin for anadditional 24 hours. H) shows cells transfected with GFP-TDP-43 andT240R, showing lack of GFP fluorescence with parkin mutant. I) showsqRT-PCR showing Park2 mRNA in M17 cells transfected with LacZ TDP-43,parkin and TDP-43+parkin. J) shows quantification of qRT-PCR showingrelative Park2 mRNA levels normalized to GADPH and expressed as %control. N=4, P<0.05, ANOVA, Neumann Keuls. K) shows qRT-PCR showingPark2 mRNA in rat cortex injected with LacZ (un-injected control),TDP-43, parkin and TDP-43+parkin. L) shows quantification of qRT-PCRshowing relative Park2 mRNA levels normalized to GADPH and expressed as% control. N=4, P<0.05, ANOVA, Neumann Keuls. M) shows qRT-PCR showingPark2 mRNA in TDP43-Tg and control cortex. N) shows quantification ofqRT-PCR showing relative Park2 mRNA levels normalized to GADPH andexpressed as % control. N=3, P<0.05, ANOVA, Neumann Keuls.

FIG. 74 is a schematic showing potential effects of parkin on TDP-43localization.

FIG. 75 shows the distribution of GFP-tagged TDP-43 in M17 cellstransfected with 3 mg cDNA for 24 hrs and then treated with Nilotinib(10 mM) or Bosutinib (5 mM) and HDAC6 inhibitor Tubacin (5 mM) foradditional 24 hrs. Inserts (B&D) represent higher magnification imagesshowing translocation of GFP-tagged TDP-43 from nucleus (A) into thecytosol (B&D, and inserts), while tubacin impairs translocation (C&E).

DETAILED DESCRIPTION

Provided herein are methods of treating or preventing aneurodegenerative disease, a myodegenerative disease or a prion disease.Neurodegenerative diseases include amyotrophic lateral sclerosis,Alzheimer's disease, frontotemporal dementia, frontotemporal dementiawith TDP-43, frontotemporal dementia linked to chromosome-17, Pick'sdisease, Parkinson's disease, Huntington's chorea, mild cognitiveimpairment, Lewy Body disease, multiple system atrophy, progressivesupranuclear palsy, and cortico-basal degeneration in a subject. Themethods include the use of tyrosine kinase inhibitors. The methods alsoinclude the use of tyrosine kinase inhibitors wherein the tyrosinekinase inhibitor is not Gleevec and wherein the tyrosine kinaseinhibitor crosses the blood brain barrier. The methods also include theuse of tyrosine kinase inhibitors, wherein the tyrosine kinaseinhibitors are not c-Abl tyrosine kinase inhibitors or are not specificc-Abl inhibitors.

Provided herein is a method of treating or preventing aneurodegenerative disease in a subject, comprising selecting a subjectwith a neurodegenerative disease of the central nervous system, amyodegenerative disease or a prion disease or at risk for aneurodegenerative disease of the central nervous system, amyodegenerative disease or a prion disease and administering to thesubject an effective amount of a tyrosine kinase inhibitor, as describedthroughout. Optionally, the tyrosine kinase inhibitor is not Gleevec andthe tyrosine kinase inhibitor crosses the blood brain barrier. Forexample, the tyrosine kinase inhibitor is selected from the groupconsisting of nilotinib, bosutinib, and a combination thereof.

In the methods provided herein, neurodegenerative diseases of thecentral nervous system include, but are not limited to, AmyotrophicLateral Sclerosis, Alzheimer's Disease, Parkinson's Disease,frontotemporal dementia, Huntington's Disease, Mild CognitiveImpairment, an α-Synucleinopathy, a Tauopathy or a pathology associatedwith intracellular accumulation of TDP-43.

In the methods provided herein, myodegenerative diseases include, butare not limited to, inclusion body myositis (IBM), spinal-bulbarmuscular atrophy (SBMA), and motor neuron disease (MND).

In the methods provided herein, prion diseases or transmissiblespongiform encephalopathies (TSEs) include, but are not limited to,Creutzfeldt-Jakob Disease (CJD), Variant Creutzfeldt-Jakob Disease(vCJD), Gerstmann-Straussler-Scheinker Syndrome, Fatal Familial Insomniaand Kuru in humans. Animal prion diseases include, but are not limitedto, Scrapie, Bovine Spongiform Encephalopathy (BSE), Chronic WastingDisease (CWD), Transmissible mink encephalopathy, Feline spongiformencephalopathy and Ungulate spongiform encephalopathy.

Examples of tyrosine kinase inhibitors include, but are not limited to,nilotinib, bosutinib, or a combination thereof. Nilotinib (or AMN-107),which is sold as TASIGNA® (Novartis, Basel Switzerland), and Bosutinib(or SKI-606) (Pfizer, New York, N.Y.) are Bcr-Abl tyrosine kinaseinhibitors developed as alternatives to the Bcr-Abl tyrosine kinaseinhibitor and CML treatment, Imatinib. Nilotinib is an Abelson kinaseinhibitor (c-Abl kinase), whereas Bosutinib is a dual Src and c-Ablkinase inhibitor. These agents are cancer therapeutics that blockcellular proliferation of cancer cells and are currently used primarilyin the treatment of chronic myelogenous leukemia (CML).

In neurodegenerative disorders, normal autophagic flux is altered,resulting in the accumulation of autophagic vacuoles or autophagosomes.This is shown in the Examples where the accumulation of vacuoles is seenin human patients with decreased parkin solubility activity. Normalautophagy is a dynamic multi-step process that prevents proteinaccumulation via sequestration into autophagic vacuoles(autophagosomes). Subsequent fusion of the autophagosomes with lysosomesresults in protein degradation. Interruption of this process results inaccumulation of protein aggregates and neurodegeneration. Parkin is anE3 ligase involved in proteasomal and autophagic degradation via proteinubiquitination and autophagosome maturation.

Tyrosine kinase inhibition activates parkin-mediated clearance ofaggregated proteins and/or activates ubiquitination. Activation ofparkin by tyrosine kinase inhibitors up-regulates protein levels ofbeclin, thus facilitating autophagic clearance. For example, nilotinib,bosutinib, or a combination thereof activates parkin-mediated clearanceof aggregated proteins and/or activates ubiquitination. Significantly,both nilotinib and bosutinib cross the blood brain barrier and promoteparkin activity in the central nervous system. Parkin activity promotesautophagic clearance of amyloid beta and alpha-synuclein and causesprotective mechanisms for parkin ubiquitination, for example,sequestration of TDP-43 associated with amyotrophic lateral sclerosis(ALS) and frontotemporal dementia. Furthermore, the tyrosine kinaseinhibitors rescue brain cells from apoptotic death in neurodegenerativedisease. In the case of ALS, the inhibitors increase ubiquitination ofTDP-43 and translocate it from the nucleus, where it interactsdeleteriously with mRNA and thousands of genes, to the cytosol where itis sequestered.

The method optionally includes selecting a subject with aneurodegenerative disease or at risk for developing a neurodegenerativedisease. One of skill in the art knows how to diagnose a subject with orat risk of developing a neurodegenerative disease. For example, one ormore of the follow tests can be used genetic test (e.g., identificationof a mutation in TDP-43 gene) or familial analysis (e.g., familyhistory), central nervous system imaging (e.g., magnetic resonanceimaging and positron emission tomography), clinical or behavioral tests(e.g., assessments of muscle weakness, tremor, or memory), laboratorytests.

The method optionally further includes administering a secondtherapeutic agent to the subject. The second therapeutic agent isselected from the group consisting of levadopa, a dopamine agonist, ananticholinergic agent, a monoamine oxidase inhibitor, a COMT inhibitor,amantadine, rivastigmine, an NMDA antagonist, a cholinesteraseinhibitor, riluzole, an anti-psychotic agent, an antidepressant, andtetrabenazine.

By way of example, provided herein is a method of treating amyotrophiclateral sclerosis or frontotemporal dementia in a subject. The methodincludes selecting a subject with amyotrophic lateral sclerosis orfrontotemporal dementia, wherein the subject has a TDP-43 pathology, andadministering to the subject an effective amount of the tyrosine kinaseinhibitor. The TDP-43 pathology can be, for example, a TDP-43 mutation.For example, the tyrosine kinase inhibitor is a tyrosine kinaseinhibitor that is not Gleevec and crosses the blood brain barrier. Inanother example, the tyrosine kinase inhibitor is selected from thegroup consisting of nilotinib, bosutinib, and a combination thereof.TDP-43 pathology occurs in ALS and frontotemporal dementia and anelevated level of TDP-43 in the cytoplasm has been noted in some casesof ALS and frontotemporal dementia. Mutations in the gene that encodesthe TDP-43 protein (known as TARDBP) have been discovered in someindividuals with ALS and frontotemporal dementia. Thus, mutated TDP-43or mutations in TARDBP can serve as biomarkers for a subject at risk forALS or frontotemporal dementia.

Also provided herein is a method of promoting parkin activity in asubject. The method includes selecting a subject with a disorderassociated with decreased parkin activity and administering to thesubject an effective amount of the tyrosine kinase inhibitor. Forexample, the tyrosine kinase inhibitor is a tyrosine kinase inhibitorthat is not Gleevec and crosses the blood brain barrier. In anotherexample, the tyrosine kinase inhibitor is selected from the groupconsisting of nilotinib, bosutinib, and a combination thereof.

Methods for measuring parkin activity are known in the art. See, forexample, Schlossmacher and Shimura (“Parkinson's disease: assays for theubiquitin ligase activity of neural Parkin,” Methods Mol. Biol. 301:351-69 (2005)); Morrison et al. (“A simple cell based assay to measureParkin activity,” J. Neurochem. 116(3): 342-9 (2011)) and Burns et al.(Hum. Mol. Genet. 18 3206-3216 (2009)).

Further provided is a method of treating or preventing aneurodegenerative disease in a subject, comprising selecting a subjectwith a neurodegenerative disease or at risk for a neurodegenerativedisease, determining that the subject has a decreased level of parkinactivity relative to a control, and administering to the subject aneffective amount of a small molecule that increases parkin activity,wherein the small molecule is not Gleevec. For example, the smallmolecule can be a tyrosinse kinase inhibitor, such as, for example, atyrosine kinase inhibitor that crosses the blood brain barrier. Thetyrosine kinase inhibitor can also be selected from the group consistingof nilotinib, bosutinib, and a combination thereof.

The term effective amount, as used throughout, is defined as any amountnecessary to produce a desired physiologic response. The effectiveamount is generally less than the amount used in chemotherapeuticmethods to treat cancer or leukemia, but is an amount sufficient toactivate parkin. Thus, the dosage of the tyrosine kinase inhibitor inthe present methods is optionally lower than a chemotherapeutic dosageof the inhibitor. For example, the dosage is optionally less than about10 mg/kg and can be 8, 7, 6, 5, 4, 3, 2, or 1 mg/kg. One of skill in theart would adjust the dosage as described below based on specificcharacteristics of the inhibitor and the subject receiving it.

Furthermore, the duration of treatment can be longer in the presentmethods than the duration of chemotherapeutic treatment, for examplecancer treatment. For example, administration to a subject with or atrisk of developing a neurodegenerative disease could be at least daily(e.g., once, twice, three times per day) for weeks, months, or years solong as the effect is sustained and side effects are manageable.

There are several ways to activate parkin. Parkin immuno-precipitationand incubation with a series of activating and ligating enzymes (E andE2) and ATP result in parkin auto-ubiquitination, and confer activity toubiquitinate targets like Abeta and TDP-43. So, in order to increaseparkin activity, parkin expression must be increased. This can beachieved by viral introduction of parkin which leads to over-expressionof the protein and increased activity. As shown in the Examples, thismethod repeatedly increases protein degradation via the proteasomeand/or autophagy. Parkin can also be activated by administration of atyrosine kinase, such as, for example, nilotinib or bosutinib, whichleads to increased levels of parkin and increased activity.

Effective amounts and schedules for administering the tyrosine kinaseinhibitor can be determined empirically and making such determinationsis within the skill in the art. The dosage ranges for administration arethose large enough to produce the desired effect in which one or moresymptoms of the disease or disorder are affected (e.g., reduced ordelayed). The dosage should not be so large as to cause substantialadverse side effects, such as unwanted cross-reactions, cell death, andthe like. Generally, the dosage will vary with the type of inhibitor,the species, age, body weight, general health, sex and diet of thesubject, the mode and time of administration, rate of excretion, drugcombination, and severity of the particular condition and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any contraindications. Dosages canvary, and can be administered in one or more dose administrations daily.

The tyrosine kinase inhibitor is administered systemically andpreferably orally.

Also provided herein is a method of inhibiting or preventing toxicprotein aggregation in a neuron and/or rescuing a neuron fromdegeneration. The method includes contacting the neuron with aneffective amount of a tyrosine kinase inhibitor. For example, thetyrosine kinase inhibitor is a tyrosine kinase inhibitor that is notGleevec and crosses the blood brain barrier. In another example, thetyrosine kinase inhibitor is selected from the group consisting ofnilotinib, bosutinib, and a combination thereof. The toxic proteinaggregate optionally comprises one or more of an amyloidogenic protein,alpha-synuclein, tau, insoluble Parkin, TDP-43, a prion protein or toxicfragments thereof. By amyloidogenic protein is meant a peptide,polypeptide, or protein that has the ability to aggregate. An example ofan amyloidogenic protein is β-amyloid.

The contacting is performed in vivo or in vitro. The in vivo method isuseful in treating a subject with or at risk of developing toxic proteinaggregates and comprises administering the tyrosine kinase inhibitor asdescribed above. The in vitro method is useful for example in treatingneural cells prior to transplantation. The tyrosine kinase inhibitor isgenerally added to a culture medium. Optionally, the target neurons arecontacted with a second therapeutic agent as described above.

Also provided herein is a method of inhibiting or preventing toxicprotein aggregation in a muscle cell and/or rescuing a muscle cell fromdegeneration. Further provided is a method of inhibiting or preventingtoxic protein aggregation in a glial cell and/or rescuing a glial cellfrom degeneration. The method includes contacting the glial cell with aneffective amount of a tyrosine kinase inhibitor. For example, thetyrosine kinase inhibitor is a tyrosine kinase inhibitor that is notGleevec and crosses the blood brain barrier.

The disclosure also provides a pharmaceutical pack or kit comprisingpackaging and/or one or more containers filled with one or more of theingredients of the pharmaceutical compositions. Instructions for use ofthe composition can also be included.

Provided herein is a pharmaceutical composition comprising an effectiveamount of the tyrosine kinase inhibitor in a pharmaceutically acceptablecarrier. The term carrier means a compound, composition, substance, orstructure that, when in combination with a compound or composition, aidsor facilitates preparation, storage, administration, delivery,effectiveness, selectivity, or any other feature of the compound orcomposition for its intended use or purpose. For example, a carrier canbe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject. Such pharmaceuticallyacceptable carriers include sterile biocompatible pharmaceuticalcarriers, including, but not limited to, saline, buffered saline,artificial cerebral spinal fluid, dextrose, and water.

Depending on the intended mode of administration, the pharmaceuticalcomposition can be in the form of solid, semi-solid, or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, aerosols, or suspensions, preferably in unit dosageform suitable for single administration of a precise dosage. Thecompositions will include a therapeutically effective amount of thecompound described herein or derivatives thereof in combination with apharmaceutically acceptable carrier and, in addition, can include othermedicinal agents, pharmaceutical agents, carriers, or diluents. Bypharmaceutically acceptable is meant a material that is not biologicallyor otherwise undesirable, which can be administered to an individualalong with the selected compound without causing unacceptable biologicaleffects or interacting in a deleterious manner with the other componentsof the pharmaceutical composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, or other materialwell known in the art for use in pharmaceutical formulations. The choiceof a carrier for use in a composition will depend upon the intendedroute of administration for the composition. The preparation ofpharmaceutically acceptable carriers and formulations containing thesematerials is described in, e.g., Remington's Pharmaceutical Sciences,21st Edition, ed. University of the Sciences in Philadelphia,Lippincott, Williams & Wilkins, Philadelphia Pa., 2005. Examples ofphysiologically acceptable carriers include buffers such as phosphatebuffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN® (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing the compound described herein orpharmaceutically acceptable salts or prodrugs thereof suitable forparenteral injection can comprise physiologically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsions,and sterile powders for reconstitution into sterile injectable solutionsor dispersions. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols(propyleneglycol, polyethyleneglycol, glycerol, and the like), suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike can also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds describedherein or pharmaceutically acceptable salts or prodrugs thereof includecapsules, tablets, pills, powders, and granules. In such solid dosageforms, the compounds described herein or derivatives thereof is admixedwith at least one inert customary excipient (or carrier) such as sodiumcitrate or dicalcium phosphate or (a) fillers or extenders, as forexample, starches, lactose, sucrose, glucose, mannitol, and silicicacid, (b) binders, as for example, carboxymethylcellulose, alignates,gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, asfor example, glycerol, (d) disintegrating agents, as for example,agar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain complex silicates, and sodium carbonate, (e) solution retarders,as for example, paraffin, (f) absorption accelerators, as for example,quaternary ammonium compounds, (g) wetting agents, as for example, cetylalcohol, and glycerol monostearate, (h) adsorbents, as for example,kaolin and bentonite, and (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, tablets, andpills, the dosage forms can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They can contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of the compounds describedherein or pharmaceutically acceptable salts or prodrugs thereof includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms can contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents, and emulsifiers, as for example,ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid estersof sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, can contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the compounds described herein or pharmaceuticallyacceptable salts or prodrugs thereof for rectal administrations areoptionally suppositories, which can be prepared by mixing the compoundswith suitable non-irritating excipients or carriers such as cocoabutter, polyethyleneglycol or a suppository wax, which are solid atordinary temperatures but liquid at body temperature and therefore, meltin the rectum or vaginal cavity and release the active component.

Throughout, treat, treating, and treatment refer to a method of reducingor delaying one or more effects or symptoms of a neurodegenerativedisease or disorder. The subject can be diagnosed with disease ordisorder. Treatment can also refer to a method of reducing theunderlying pathology rather than just the symptoms. The effect of theadministration to the subject can have the effect of but is not limitedto reducing one or more symptoms of the neurodegenerative disease ordisorder, a reduction in the severity of the neurological disease orinjury, the complete ablation of the neurological disease or injury, ora delay in the onset or worsening of one or more symptoms. For example,a disclosed method is considered to be a treatment if there is about a10% reduction in one or more symptoms of the disease in a subject whencompared to the subject prior to treatment or when compared to a controlsubject or control value. Thus, the reduction can be about a 10, 20, 30,40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between.

As utilized herein, by prevent, preventing, or prevention is meant amethod of precluding, delaying, averting, obviating, forestalling,stopping, or hindering the onset, incidence, severity, or recurrence ofthe neurodegenerative disease or disorder. For example, the disclosedmethod is considered to be a prevention if there is a reduction or delayin onset, incidence, severity, or recurrence of neurodegeneration or oneor more symptoms of neurodegeneration (e.g., tremor, weakness, memoryloss, rigidity, spasticity, atrophy) in a subject susceptible toneurodegeneration as compared to control subjects susceptible toneurodegeneration that did not receive an agent that activates parkin.The disclosed method is also considered to be a prevention if there is areduction or delay in onset, incidence, severity, or recurrence ofneurodegeneration or one or more symptoms of neurodegeneration in asubject susceptible to neurodegeneration after receiving an agent thatpromotes parkin activity as compared to the subject's progression priorto receiving treatment. Thus, the reduction or delay in onset,incidence, severity, or recurrence of neurodegeneration can be about a10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction inbetween.

As used throughout, by subject is meant an individual. Preferably, thesubject is a mammal such as a primate, and, more preferably, a human.Non-human primates are subjects as well. The term subject includesdomesticated animals, such as cats, dogs, etc., livestock (for example,cattle, horses, pigs, sheep, goats, etc.) and laboratory animals (forexample, ferret, chinchilla, mouse, rabbit, rat, gerbil, guinea pig,etc.). Thus, veterinary uses and medical formulations are contemplatedherein.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including inthe method are discussed, each and every combination and permutation ofthe method, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties. Anumber of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

EXAMPLES Example 1

Methods for the animal experiments described herein are detailed inExamples 2-5. Cell culture experiments are referenced below andexplained in Burns et al. (Human Molecular Genetics. 2009) and Rebeck etal. (J. Biol. Chem. 2010, 285:7440-7446). Additional details areprovided in the brief description of the figures. Using these methods,cellular mechanisms (FIG. 1) associated with parkin activity inneurodegenerative conditions and upon intervention with tyrosine kinaseinhibitors were studied. These studies revealed that tyrosine kinaseinhibition activates parkin and increases its interaction with beclin-1,resulting in maturation of phagophores into phagosomes and clearance(FIG. 2). It was also shown that parkin interacts with beclin-1 in wildtype, but not parkin −/−mice (FIG. 3). As shown in FIGS. 4-5, 3×APP micetreated with either Nilotinib or Bosutinib resulted in reduced Aβ₁₋₄₂ inthe brain lysates of these mice as compared to treatment with DMSO.Also, as shown in FIGS. 6-8, treatment of A53T mice (A53T-Tg) withBosutinib at different dosages and dosage schedules resulted in adecrease in human α-synuclein in the brain lysates of these mice, ascompared to treatment with DMSO. Further, as shown in FIGS. 9-10,treatment of A53T mice (A53T-Tg) with Nilotinib at different dosages anddosage schedules resulted in a decrease in human α-synuclein in thebrain lysates of these mice, as compared to treatment with DMSO.Decreases in human soluble Aβ₁₋₄₂ and human soluble Aβ₁₋₄₀ in the brainlysates of triple mutant APP-AD mice were also observed after treatmentwith Bosutinib (FIGS. 11A and B). Treatment with Bosutinib also resultedin increased parkin levels and decreased levels of phosphorylated Tau(FIGS. 11C and D).

In other experiments, M17 cells transfected with Tau cDNA were treatedwith Nilobinib and Tubacin (an HDAC6 inhibitor). Treatment withNilotinib resulted in a decrease in human Tau, a decrease in humanAβ₁₋₄₂ and a decrease in α-synuclein as compared to transfected cells.

Treatment of lentiviral Aβ₁₋₄₂ injected mice with bosutinib alsoresulted in decreased levels of Aβ₁₋₄₂ in brain lysates (FIG. 12).

In another experiment, mice were injected stereotaxically (bilaterally)with lentiviral α-synuclein into the substantia nigra for 3 weeks. Then,half of the animals were injected with 10 mg/Kg nilotinib and the otherhalf with DMSO. The effects of α-synuclein expression and tyrosinekinase inhibition on brain (FIG. 13A) and blood (FIG. 13B) levels ofα-synuclein were compared. As shown in FIG. 13, α-synuclein expressionin the brain increases its blood level and tyrosine kinase inhibitionreverses these effects in a parkin-dependent manner.

In another experiment, mice were injected stereotaxically (bilaterally)with lentiviral α-synuclein into the substantia nigra for 3 weeks. Then,half of the animals were injected with 5 mg/Kg Bosutinib and the otherhalf with DMSO. The effects of α-synuclein expression and tyrosinekinase inhibition on brain (FIG. 14A) and blood (FIG. 14B) levels ofα-synuclein were compared. As shown in FIG. 14, α-synuclein expressionin the brain increases its blood level and tyrosine kinase inhibitionreverses these effects in a parkin-dependent manner.

In another study, mice were injected stereotaxically (bilaterally) withlentiviral α-synuclein into the substantia nigra for 3 weeks. Then halfthe animals were injected with 10 mg/kg Nilotinib or 5 mg/Kg Bosutiniband the other half with DMSO. As shown in FIG. 185, the effects ofα-synuclein expression and tyrosine kinase inhibition on dopamine andhomovanillic acid (HVA) levels (ELISA) were compared. The effects oftreatment on motor performance were evaluated using rotarod (FIG. 15B).This study shows that α-synuclein induced loss of dopamine andhomovanillic acid (HVA) levels. Tyrosine kinase inhibition reversedthese effects and improved motor performance.

In another study, transgenic A53T mice that express human α-synucleinthroughout the brain (excluding substantia nigra) were injected with 10mg/kg Nilotinib or 5 mg/Kg Bosutinib or DMSO once daily for 3 weeks. Theeffects of α-synuclein expression and tyrosine kinase inhibition ondopamine and homovanillic acid (HVA) levels (ELISA) were compared. Theeffects of treatment on motor performance were tested using rotarod.α-synuclein did not induce loss of Dopamine and HVA (due to absence ofα-synuclein expression in dopamine producing neurons in these mice.Tyrosine kinase inhibition increased dopamine and HVA. Motor performancealso increased.

Studies were also performed to show that Aβ₁₋₄₂ and Aβ₁₋₄₀ accumulate inAV-10 in Tg-APP animals, but drug treatment enhances autophagicclearance via deposition of Aβ₁₋₄₂ or Aβ₁₋₄₀, respectively, in AV-20 andlysosomes (See FIGS. 16 and 17, respectively). Additional studies showsthat p-Tau and parkin also accumulate in AV-10 in Tg-APP animals, butdrug treatment enhances autophagic clearance via deposition of p-Tau orparkin in AV-20 and lysosomes (See FIGS. 18 and 19, respectively).

In another study, it was shown that Aβ₁₋₄₂ and p-Tau at serine 396accumulate in the brains of mice injected with lentiviral Aβ₁₋₄₂, butdrug treatment enhances autophagic clearance via deposition of Aβ₁₋₄₂ orp-Tau in AV-20 and lysosomes (See FIGS. 20 and 21, respectively). Alsoshown is that p-Tau and α-synuclein accumulate in the brains of miceinjected with lentiviral α-synuclein, but drug treatment enhancesautophagic clearance via deposition of p-Tau or α-synuclein in AV-20 andlysosomes (See FIGS. 22 and 23, respectively). Further shown is thatα-synuclein and p-Tau accumulate in AV-10 of A53T brains, but drugtreatment enhances autophagic clearance via deposition of p-Tau orα-synuclein in AV-20 and lysosomes (See FIGS. 24 and 25, respectively).Parkin also accumulates in the brains of A53T mice, but as shown in FIG.26, drug treatment enhances autophagic clearance via deposition ofparkin in AV-20 and lysosomes. As shown in FIG. 27, tyrosine kinaseinhibition increases parkin activity and facilitates autophagicclearance of p-Tau. This process requires Tau stabilization of intactmicrotubules. Tyrosine kinase activation, p-Tau accumulation andimpaired autophagy are recognized in neurodegeneration. Decreased parkinsolubility and accumulation with intracellular Aβ and p-Tau inautophagic vacuoles in AD brains occurs, while exogenous parkinfacilitates autophagic clearance in animal models.

In another study, wild type or parkin−/− mice were injected withlentiviral Tau±Aβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kg Nilotinibor DMSO once a day for 3 (additional) consecutive weeks. Brain tissueswere fractionated to isolate AVs and human specific ELISA was performedto determine Aβ₁₋₄₂ contents. Aβ₁₋₄₂ accumulates in AV-10 in lentivirusinjected brains but drug treatment enhances autophagic clearance viadeposition of Aβ₁₋₄₂ in AV-20 and lysosome. It was also observed thatautophagic clearance is parkin-dependent. Further, this study shows thatTau expression leads to Aβ₁₋₄₂ accumulation in AV10 and AV20, but not inlysosomes, indicating decreased fusion between autophagosomes andlysosomes.

In another study, wild type or Tau−/− mice were injected with lentiviralAβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kg Nilotinib or 5 mg/KgBosutinib or DMSO once a day for 3 (additional) consecutive weeks. Braintissues were fractionated to isolate AVs and human specific ELISA wasperformed to determine protein contents. Results showed that Aβ₁₋₄₂accumulates in AV-10 in lentivirus injected brains but drug treatmentenhances autophagic clearance via deposition of Aβ₁₋₄₂ in AV-20 andlysosomes. Autophagic clearance is less efficient in Tau null animalswith Aβ₁₋₄₂ accumulation in AV-10 and AV-20.

In another study, wild type or parkin−/− mice injected with lentiviralhuman Tau±Aβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kg Nilotinib or30 μL DMSO once a day for 3 (additional) consecutive weeks. Braintissues were fractionated to isolate AVs and mouse specific ELISA wasperformed to determine protein contents. Results showed that P-Tau atserine 396 accumulates in AV-10 in lentivirus injected brains but drugtreatment enhances autophagic clearance via deposition of p-Tau in AV-20and lysosomes, where it is degraded.

In another study, wild type or parkin−/− mice were injected withlentiviral Tau±Aβ₁₋₄₂ for 3 weeks and treated IP with 10 mg/kg Nilotinibor 5 mg/Kg Bosutinib or DMSO once a day for 3 (additional) consecutiveweeks. Brain tissues were fractionated to isolate AVs and human specificELISA was performed to determine protein contents. Results showed thatP-Tau at serine 396 accumulates in AV-10 in lentivirus injected brains,but drug treatment enhances autophagic clearance via deposition of p-Tauin AV-20 and lysosomes, where it is degraded.

FIG. 28 shows A) phosphorylated c-Abl at tyrosine 412 (T412) and B)endogenous parkin expression merged in C) hippocampus of 6 month oldC57BL/6 mice treated IP with DMSO daily for 3 weeks. FIG. 28 also showsD) decreased phosphorylated c-Abl at tyrosine 412 (T412) and E)increased endogenous parkin expression merged in F) hippocampus of 6month old C57BL/6 mice treated IP with 5 mg/kg Bosutinib daily for 3weeks.

FIG. 29 shows A) parkin and B) Aβ expression merged in C) cortex of 6months old Tg-APP mice treated with DMSO or 5 mg/kg Bosutinib (D-F) oncea day for 3 weeks. Using a different combination of antibodies (seefigure G-I show expression of parkin (G) and Aβ (H) in the hippocampusof Tg-APP mice treated DMSO. J-H show the increase in parkin level inanimals treated for 3 weeks once a day with Bosutinib (J) along withdecreased plaque levels (K and L) in the hippocampus.

FIG. 30 shows plaque Aβ stained with 6E10 antibody and counterstainedwith DAB in the brain of Tg-APP animals treated IP with DMSO once a dayfor 3 weeks.

FIG. 31 shows plaque Aβ stained with 6E10 antibody and counterstainedwith DAB in the brain of Tg-APP animals treated IP with 5 mg/kgBosutinib once a day for 3 weeks. A decrease in plaque formation in theanimals treated with Bosutinib as compared to the animals treated withDMSO was observed.

FIG. 32 shows that Bosutinib decreases α-synuclein levels in transgenicmice expressing A53T throughout the brain. FIGS. 32A-D show humanα-synuclein expression in lentiviral LacZ injected (for 3 weeks)substantia nigra with A) DMSO and B) 5 mg/kg Bosutinib once a day for 3weeks. C and D show human α-synuclein expression in lentiviralα-synuclein injected (for 3 weeks) substantia nigra with C) DMSO and D)or Bosutinib once a day for 3 weeks. FIGS. 32E-H show tyrosinehydroxylase (TH) expression in lentiviral LacZ injected (for 3 weeks)substantia nigra with E) DMSO and F) 5 mg/kg Bosutinib once a day for 3weeks. G and H show TH expression in lentiviral α-synuclein injected(for 3 weeks) substantia nigra with G) DMSO and H) or Bosutinib once aday for 3 weeks. synuclein decreases TH neurons and Bosutinib rescuesthese cells. FIGS. 32I-L show human α-synuclein expression in A53T micein I) Cortex, J) Striatum, K) Brainstem and L) Hippocampus treated withDMSO for 3 weeks. FIGS. 32M-P show human α-synuclein expression in A53Tmice in M) cortex, N) striatum, O) brainstem and P) hippocampus treatedwith 5 mg/kg Bosutinib for 3 weeks.

Performance tests were also done. As shown in FIGS. 33A and B, IPtreatment with 5 mg/kg Bosutinib once daily for 3 weeks improvedcognitive behavior in mice injected bilaterally with lentiviral Aβ₁₋₄₂for 3 weeks prior to drug treatment. Bosutinib treated mice found theplatform (A) but DMSO treated mice spent more time in NW area, wherethey were initially placed or the NE or SW area, without effectivelyfinding platform area. Bosutninb improved cognitive performance in aparkin-dependent manner as the parkin−/− mice did not seem to learnmuch. FIG. 41B shows that Bosutinib treated mice traveled less distancewith less speed but entered the platform area more than DMSO treatedmice.

Studies also showed that parkin activity was increased in human M17neuroblastoma cells after treatment with Nilotinib or Bosutinib (FIG.34A). Treatment with Nilotinib also resulted in increased parkin levelsin the brain lysates of wild type mice injected with lentivirala-synuclein prior to treatment (FIG. 34B).

Western blot analysis of brain lysates from wild type mice treated withBosutinib revealed that Bosutinib boosts autophagy and degradesubiquitinated proteins. Western blot analysis of brain lysates fromTg-APP mice treated with 5 mg/kg Bosutinib for 3 additional weeks showeddecreased levels of c-Abl, increased parkin and alteration of differentmolecular markers of autophagy, indicating that Aβ alters normalautophagy and Bosutinib boosts autophagy to clear Aβ₁₋₄₂ (FIG. 35).Western blot analysis of brain lysates from Tg-APP mice treated withBosutinib showed alterations in the levels of molecular markers ofautophagy (FIG. 36). Western blot analysis of brain lysates from Tg-APPmice treated with Bosutinib also showed decreased levels of C-terminalfragments (CTFs) and phosphor-tyrosine (FIG. 37).

Western blot analysis of brain lysates from Tg-APP mice treated with 5mg/kg Bosutinib once a day for additional weeks showed decreased levelsof different Tau isotopes (FIG. 38). Western blot analysis of brainlysates from wild type mice expressing lentiviral Aβ₁₋₄₂ (3 weeks) withand without Bosutinib (5 mg/kg) treatment for 3 additional weeks, showeddecreased c-Abl and increased parkin levels with Bosutinib treatment,indicating that Aβ₁₋₄₂ activates c-Abl and Bosutinib activates parkin.

Western blot analysis of brain lysates from wild type mice expressinglentiviral Aβ₁₋₄₂ (3 weeks) with and without Bosutinib (5 mg/kg)treatment for 3 additional weeks showed levels of different molecularmarkers of autophagy, indicating that Aβ₁₋₄₂ alters normal autophagy andBosutinib boosts autophagy to clear Aβ₁₋₄₂ (FIG. 39). Western blotanalysis of brain lysates from wild type mice expressing lentiviralAβ₁₋₄₂ (3 weeks) with and without Bosutinib treatment for 3 additionalweeks, showed decreased levels of ubiquitin (top blot) and panphospho-tyrosine (second blot) and SIAH2, indicating that Bosutinib is abroad tyrosine kinase inhibitor (FIG. 40).

Western blot analysis of brain lysates from wild type mice expressinglentiviral Aβ₁₋₄₂ (3 weeks) with and without Bosutinib treatment for 3additional weeks showed decreased levels of different Tau isotopes (FIG.41). Western blot analysis of brain lysates from wild type miceexpressing lentiviral α-synuclein (3 weeks) with and without Bosutinibtreatment for 3 additional weeks was also performed. This blots showincreased α-synuclein in lentiviral synuclein injected animals, alongwith decreased c-Abl levels and phosphorylation, increased parkin levelsand markers of autophagy, including P62, HDAC6, LC3 and ATG12 comparedto loading controls tubulin and MAP2 (FIG. 42).

Example 2: Parkin Inactivation in Parkinson's Disease

To determine the role of parkin and its association with baselineautophagy in sporadic PD, human postmortem nigrostriatal tissues wereanalyzed via fractionation to determine protein solubility and theeffects of parkin on autophagic clearance in lentiviral gene transferanimal models were investigated. Whether lentiviral expression ofα-Synuclein affects autophagy and if parkin activity reversesα-Synuclein effects was investigated. Animal models expressinglentiviral α-Synuclein were studied and it was found that parkinexpression decreases α-Synuclein levels in the absence ofubiquitination. Whether parkin expression regulates α-Synucleinclearance via autophagic degradation was studied.

Human postmortem brain tissues. Human postmortem caudate and midbrainregions from 22 PD patients and 15 age matched control subjects wereobtained from John's Hopkins University brain bank. The age, sex, stageof disease and postmortem dissection (PMD) are summarized for eachpatient in Table 1 and 2. The cause of death is not known. To extractthe soluble fraction of proteins, 0.5 g of frozen brain tissues werehomogenized in 1×STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mMEDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease and phosphatasecocktail inhibitor), centrifuged at 10,000 g for 20 min at 4° C., andthe supernatants were collected. All samples were then analyzed by ELISA(see below) or Western blot using 30 μg of protein. To extract theinsoluble fraction, the pellet was re-suspended in 4M urea solution andcentrifuged at 10,000 g for 15 min, and the supernatant was collectedand 30 μg of protein was analyzed by Western blot. Western blots werequantified by densitometry using Quantity One 4.6.3 software (Bio-Rad,Hercules, Calif.). Densitometry was obtained as arbitrary numbersmeasuring band intensity. Data were analyzed as mean±Standard deviation,using Two-tailed t-test (P<0.02) and ANOVA, Neumann Keuls with multiplecomparisons (P<0.05) to compare PD and control groups.

Immunohistochemistry on slides from human patients was performed on 30μm thick paraffin embedded brain slices de-paraffinized in Xylenes 2×5minutes and sequential ethanol concentration, blocked for 1 hour in 10%horse serum and incubated overnight with primary antibodies at 4° C.After 3×10 minute washes in 1×PBS, the samples were incubated with thesecondary antibodies for 1 hr at RT, washed 3×10 minutes in 1×PBS.Parkin was immunoprobed (1:200) with mouse anti-parkin (PRK8) antibodythat recognizes a.a. 399-465 (Signet Labs, Dedham, Mass.) or rabbitpolyclonal (1:200) anti-parkin (AB5112) antibody that recognizes a.a.305-323 (Millipore) and counterstained with DAPI. Map 2 was probed(1:300) with mouse monoclonal antibody (Pierce). Glial Fibrillary AcidProtein (GFAP) was probed (1:200) with mouse (GA5) Mouse mAb #3670 (CellSignaling) or (1:200) rabbit polyclonal (ab4674) antibody (Abcam).Tyrosine Hydroxylase (TH) was probed (1:100) with rabbit polyclonal(AB152) antibody (Millipore) and counterstained with DAB.

Stereotaxic Injection.

Lentiviral constructs were used to generate the animal models asexplained in Burns et al. (Hum. Mol. Genetics 18: 3206-3216 (2009);Khandelwal et al. (Mol. Neurodegener. 5: 47 (2010) and Herman and Moussa(Autophagy 7:919-921 (2011). Stereotaxic surgery was performed to injectthe lentiviral constructs into the striatum of 2-month old maleSprague-Dawley rats. N=8 animals were used in each treatment. A total of116 animals were used in these studies. All procedures were approved bythe Georgetown University Animal Care and Use Committee (GUACUC).

Western Blot Analysis.

To extract the soluble protein fraction, brain tissues were homogenizedin 1×STEN buffer, centrifuged at 10,000×g for 20 min at 4° C., and thesupernatants containing the soluble fraction of proteins were collected.To extract the insoluble fraction the pellet was re-suspended in 4M ureaor 30% formic acid and adjusted to pH 7 with 1N NaOH and centrifuged at10,000×g for 20 min at 4° C., and the supernatant containing theinsoluble fraction was collected and analyzed by Western blot. Totalparkin was immunoprobed (1:1000) with PRK8 antibody as indicated (Burnset al., 2009) and phospho-parkin was probed (1:1000) with anti-Ser 378antibodies (Pierce). α-Synuclein was probed with rabbit monoclonal(1:1000) antibody (Santa Cruz). Autophagy antibodies, including beclin-1(1:1000), autophagy like gene (Atg)-7 (1:1000), Atg12 (1:1000) and LC3-B(1:1000), were used to probe according to autophagy antibody sampler kit4445 (Cell Signaling, Inc). Histone deacetylase 6 (HDAC6) was probed(1:500) using rabbit polyclonal anti-HDAC6 (Abcam). Rabbit polyclonalanti-SQSTM1/p62 (Cell Signaling Technology) was used (1:500). A rabbitpolyclonal (Pierce) anti-LC3 (1:1000) and rabbit polyclonal (ThermoScientific) anti-actin (1:1000) were used. LAMP-3 was probed (1:500)rabbit polyclonal antibody (Aviva Systems). Rabbit anti-ubiquitin (SantaCruz Biotechnology) antibody (1:1000) was used. Mitochondrial proteinCOX-IV was probed (1:1000) with rabbit polyclonal (ab16056) antibody(Abcam) and human poly ADP-ribose polymerase (PARP-1) was probed(1:1500) with monoclonal (MA3-950) antibody (Pierce).

Immunohistochemistry—

These methods were performed on 20 micron-thick 4% paraformaldehyde(PFA) fixed striatal rat brain sections and compared between treatments.Parkin was probed (1:200) with Rabbit polyclonal antibody (Chemicon).Rabbit polyclonal LC3-B (1:100) was used to probe LC3-B (Cell Signaling,Inc). Thioflavin-S and nuclear DAPI staining were performed according tomanufacturer's instructions (Sigma). Stereological methods—were appliedby a blinded investigator using unbiased stereology analysis(Stereologer, Systems Planning and Analysis, Chester, Md.) to determinethe total positive cell counts in 20 cortical fields on at least 10brain sections (˜400 positive cells per animal) as indicated in (Burnset al., 2009, Khandelwal et al., 2010, Herman and Moussa, 2011).

α-Synuclein, Parkin and p-Tau Enzyme-Linked Immunosorbent Assay (ELISA)—

Specific ELISA (Invitrogen) were performed using 50 μl (1 μg/μl) ofbrain lysates detected with 50 μl primary antibody (3 h) and 100 μlanti-rabbit secondary antibody (30 min) at RT. Parkin levels usingspecific human ELISA (MYBioSource), and p-Tau and α-Synuclein levelswere measured using human specific ELISA (Invitrogen) according tomanufacturers' protocols.

Subcellular Fractionation to Isolate Autophagic Vacuoles—

0.5 g of Frozen human or animal brains were homogenized at low speed(Cole-Palmer homogenizer, LabGen 7, 115 Vac) in 1×STEN buffer andcentrifuged at 1,000 g for 10 minutes to isolate the supernatant fromthe pellet. The pellet was re-suspended in 1×STEN buffer and centrifugedonce to increase the recovery of lysosomes. The pooled supernatants werethen centrifuged at 100,000 rpm for 1 hour at 4° C. to extract thepellet containing autophagic vacuoles (AVs) and lysosomes. The pelletwas then re-suspended in 10 ml (0.33 g/ml) 50% Metrizamide and 10 ml incellulose nitrate tubes. A discontinuous Metrizamide gradient wasconstructed in layers from bottom to top as follows: 6 ml of pelletsuspension, 10 ml of 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10%Metrizamide (Marzella et al., 1982). After centrifugation at 10,000 rpmfor 1 hour at 4° C., the fraction floating on the 10% layer (Lysosome)and the fractions banding at the 24%/20% (AV 20) and the 20%/10% (AV10)Metrizamide inter-phases were collected by a syringe and examined.

Transmission Electron Microscopy—

Brain tissue were fixed in (1:4, v:v) 4% paraformaldehyde-picric acidsolution and 25% glutaraldehyde overnight, and then washed 3× in 0.1Mcacodylate buffer and osmicated in 1% osmium tetroxide/1.5% potassiumferrocyanide for 3 h, followed by another 3× wash in distilled water.Samples were treated with 1% uranyl acetate in maleate buffer for 1 h,washed 3× in maleate buffer (pH 5.2), then exposed to a graded coldethanol series up to 100% and ending with a propylene oxide treatment.Samples are embedded in pure plastic and incubated at 60° C. for 1-2days. Blocks are sectioned on a Leica ultracut microtome at 95 nm,picked up onto 100 nm formvar-coated copper grids, and analyzed using aPhilips Technai Spirit transmission EM. All sections were acquired andanalyzed by a blind investigator.

Results

Decreased Parkin Solubility in Postmortem Striatum of Sporadic PDPatients.

To determine the role of parkin in the brain of sporadic PD patients,human postmortem striatal (caudate) tissues from 12 PD patients and 7age-matched controls as described in Table 1 were analyzed. ELISAmeasurement of soluble human parkin revealed a significant (P<0.05)decrease (36%) in parkin levels in PD caudate/striatum compared tocontrol (FIG. 43A). Western blot analysis of soluble striatal extractsconfirmed the decrease in parkin levels in PD patients compared tocontrol (FIGS. 43B and C, 54%). No differences in parkin levels weredetected in PD cortex. Probing with anti-ubiquitin antibody showed ahigher smear of ubiquitinated proteins in PD striatum compared tocontrol (FIG. 43B). However, all samples with PMD greater than 16 hshowed significantly (P<0.02, two-tailed t-test) higher levels ofubiquitin (48%) in both groups and higher parkin levels (25%) with PMDgreater than 13 h within the PD group. To further investigate whetherthe decreased degradation of proteins results in alteration ofsolubility, the insoluble proteins were extracted in 4M urea. Anincrease in the level of parkin was detected in the insoluble fraction(FIGS. 43D and E, 82%) in contrast to the soluble extract, which washardly detected. Parkin phosphorylation at serine 378, which was notdetected in the soluble fraction, was observed in the insoluble extract(FIGS. 43D and E, 114%). Additionally, more ubiquitinated proteins (FIG.43D, 3rd blot) were also detected in the insoluble fraction. Thevariations among the samples are represented to show variation amongindividual samples, including soluble, insoluble and phospho-parkin(FIG. 43F). Taken together these data show decreased parkin solubilityand increased phosphorylation in PD.

Altered Parkin Expression and Loss of Tyrosine Hydroxylase Neurons inthe Nigrostriatum of Sporadic PD Patients.

To determine whether parkin expression is altered in sporadic PD, humanpostmortem midbrain sections from 10 PD patients and 8 control subjectsas identified in Table 2 were examined. To determine the difference inparkin staining between PD and control brains, serial brain sectionscollected from each case were probed with human anti-parkin antibody(PRK8) that recognizes a.a. 399-495 and counterstained with either GFAPor DAPI. Confocal microscopy was used and diffuse parkin cytosolicstaining was observed in the caudate (FIG. 44A) and within GFAP-stainedastrocytes of control brain sections (FIG. 44B), and TH staining (FIG.54C) was also observed in the caudate of a control subject (case 1683).However, intense cytosolic staining in the caudate (FIG. 44D, arrow),and within astrocytes (FIG. 44E), with diminished TH staining (FIG. 44F)were observed in a PD/AD patient (case 2215). To ascertain that parkinor GFAβ staining were not due to auto-fluorescence in human slides, theslides were incubated with and without secondary and or primaryantibodies and the absence of non-specific antibody binding wasdetermined via confocal microscopy. Parkin expression was furtherexamined in midbrain/SN brain regions. Diffuse parkin cytosolic staining(FIG. 44G) and within GFAP-stained astrocytes (FIG. 44H) with THstaining (FIG. 44I) were observed in serial sections of midbrain/SN ofcontrol brain (case 1855). Intense cytosolic parkin staining (FIG. 54J,arrow), and within astrocytes (FIG. 44K), with significantly diminishedTH staining (FIG. 44L) were observed in a PD patient (case 2315).Another combination of antibodies using the AB5112 clone that detectsparkin at a.a. 305-323 and GFAβ antibodies was used to verify theseresults. Intense cytosolic parkin staining (FIG. 44M, arrow), and withinastrocytes (FIG. 44N), with significantly diminished TH staining (FIG.44O) were observed in a PD/dementia patient (case 2243). MAP-2 was usedas a neuronal marker and co-stained with parkin (DAPI counterstain) andTH. Parkin staining (FIG. 45A) was diffuse within the cytosol and waslargely localized to MAP-2 labeled neurons (FIGS. 45B & C) in themidbrain/SN of a control subject (case 1277). TH staining was alsodetected in serial brain sections (FIG. 45D). However, more intense andless diffuse parkin staining was detected in the cytosol of DAPI stainedcells (FIG. 45E) and parkin staining was localized to MAP-2 stainedneurons (FIGS. 45F&G), with significantly decreased TH staining (FIG.45H) in the midbrain/SN of a PD/Dementia patient (case 2267).

Alteration of Baseline Autophagy in Post-Mortem Striatum of PD Patients.

To determine whether the change in parkin solubility is associated withchanges of baseline autophagy, the level of some autophagic markers inhuman PD striatal extracts was examined. The markers of the autophagiccascade were examined, including microtubule-associated light chainprotein 3 (LC3). Probing with anti-LC3 antibody suggested an increase inLC3-II levels compared to LC3-I (FIGS. 45I&J, 1^(st) blot, 78%, N=12 PDand 7 control), indicating possible conversion and lipidation of LC3.LC3-I is abundant and stable in the brain, the ratio of LC3-II to LC3-Ior the amount of LC3-II can be used to monitor the amount ofautophagosome. LC3 is expressed as three isoforms in mammalian cells,LC3-A, LC3-B and LC3-C. Because LC3-II itself is degraded by autophagythe amount of LC3 was measured using an antibody specific for the LC3-Bisoform. An increase in the level of LC3-B was detected in humanstriatal extracts from PD patients (N=12) compared to control (N=7)subjects (FIGS. 45I&J, 2^(nd) blot, 48%. P<0.05, ANOVA, Neumann Keuls).Subcellular fractionation was performed to isolate autophagic vacuolesand lysosomes the levels of α-Synuclein, parkin and p-Tau were measuredusing quantitative ELISA. First it was determined whether thesubcellular fractionation assay successfully extracted autophagosomesfrom lysosomes in frozen human tissues. Western blot analysis on PDpatients brain lysates showed the lysosome-associated membraneglycoprotein 3 (LAMP-3) in the floating fraction containing lysosomes(FIG. 45K, 1^(st) blot), while both the AV-10 and AV-20 fractionscontained LC3-B (FIG. 45K, 2^(nd) blot), suggesting that frozen humanbrains contain autophagic vacuoles and our fractionation did isolateautophagosomes from lysosomes. Probing for mitochondrial markercytochrome c oxidase-IV (COX-IV, FIG. 45K, 3^(rd) blot) and nuclearmarker Poly ADP-ribose polymerase (PARP-1, FIG. 45K, 4^(th) blot) wasalso performed, and markers were detected in all fractions, suggestingthat brain samples contained intact organelles. A comprehensive assaythat clearly shows mitochondria in autophagosomes or lysosomes must beperformed with both IHC co-labeling with LC3-COX-IV (autophagosome) orcathepsin-D-COX-IV (Lysosome) coupled with immuno-EM to determinemitochondrial accumulation in separate autophagic vacuoles. An ELISA wasused to measure protein levels in subcellular extracts. The level ofα-Synuclein was significantly increased (P<0.05, N=12 PD and 7 control)in AV-10 (31%) and AV-20 (64%) compared to control (FIG. 45L, ANOVA,Neumann Keuls), but no α-Synuclein was detected in the lysosomalfraction. Interestingly, ELISA measurement of parkin levels also showeda significant increase in AV-10 (FIG. 45M, 24%) and AV-20 (FIG. 45M,23%) and a slight non-significant (9%) increase in the lysosome in PD(N=12) compared to control (N=7) subjects. The levels of p-Tau weremeasured as another protein marker that is occasionally associated withPD pathology. Similarly, no p-Tau was detected in the lysosome but thelevels of p-Tau were significantly increased in AV-10 (54%) and AV-20(64%) compared to control (FIG. 45N, N=12 PD and 7 control). These datashow accumulation of un-degraded proteins in autophagosomes in PD.

Parkin Attenuates α-Synuclein-Induced Protein Accumulation in theStriatum.

Because increased parkin insolubility and decreased soluble parkinlevels were observed in association with alteration of autophagy in PDstriatum, it was sought to over-express parkin and determine whetherfunctional parkin can reverse α-Synuclein effects on autophagicclearance. A gene transfer animal model targeting α-Synuclein expressionto the striatum of 2-month old rats was used. Lentiviral parkin led tosignificant increases (FIG. 46A, 53% by densitometry, N=8, P<0.05) inparkin levels and lentiviral α-Synuclein led to significant increases(41%) in α-Synuclein levels. Co-expression of parkin with α-Synucleinattenuated the levels of monomeric α-Synuclein (FIG. 46A) and reducedthe level of higher molecular weight proteins back to control (LacZ) 4weeks post injection (Khandelwal et al., 2010). No changes in totalparkin levels were observed in brains injected with lentiviralα-Synuclein (FIG. 46A, 1^(st) blot) and no-phosphorylated parkin wasdetected in rat brains. Independent studies were performed to confirmchanges in α-Synuclein levels using quantitative ELISA specific forhuman α-Synuclein. The levels of human α-Synuclein were significantlyincreased (FIG. 46B, 54%, N=8) in the striatum of animals injected withlentiviral α-Synuclein compared to LacZ or parkin. Co-injection withlentiviral α-Synuclein and parkin reversed the levels of humanα-Synuclein back to control. Lentiviral delivery of parkin into thestriatum resulted in a significant increase in parkin when it wasexpressed alone (FIG. 46C, 44%, N=8) or in the presence of α-Synuclein(53%, N=8).

Changes in rat p-Tau were determined using ELISA. Expression of humanα-Synuclein leads to a significant increase (FIG. 46D, 34%, N=8) inp-Tau in the rat striatum, but co-expression of parkin reverses p-Tauback to control. Lentiviral expression of α-Synuclein in the striatumleads to detection of thioflavin-S positive staining (FIG. 46F),compared to lentiviral parkin alone (FIG. 46F). However, co-expressionof parkin with α-Synuclein prevents the appearance of thioflavin-Spositive staining (FIG. 46G); suggesting that parkin attenuation ofα-Synuclein levels can eliminate thioflavin-S positive species in thisanimal model. To ascertain that thioflavin-S staining is associated withα-Synuclein expression, striatal sections were stained with humanα-Synuclein antibody and showed no α-Synuclein staining in sections cutserially with the thioflavin-S sections from lentiviral parkin injectedrats (FIG. 46K), compared to an abundant level of α-Synuclein inlentiviral α-Synuclein injected rats, congruent with thioflavin-Sstaining (FIG. 46L), while parkin co-expression led to disappearance ofhuman α-Synuclein in the rat striatum (FIG. 46M).

Wild Type Functional Parkin, not Mutant T240R, Mediates Clearance ofα-Synuclein-Induced Autophagic Vacuoles.

It was sought to determine whether α-Synuclein expression can changenormal autophagy, leading to formation of autophagic vacuoles in vivo.EM images of striatal sections showed no vacuoles in lentiviral LacZinjected animals (FIG. 47A) 4 weeks post injection. Lentiviralexpression of α-Synuclein led to cytosolic accumulation of vacuoles(FIG. 47B, asterisks), suggesting that α-Synuclein expression altersautophagy in the rat striatum. Co-expression of parkin with α-Synucleinled to formation of autophagic vacuoles containing debris (FIG. 47C). Toascertain whether parkin function mediates clearance of autophagicvacuoles, non-functional T240R parkin was used, which is a mutant formthat loses its E3 ubiquitin ligase activity, leading to ARJPD.Co-expression of mutant T240R parkin with α-Synuclein did not preventthe accumulation of cytosolic vacuoles (FIG. 47D, asterisks), suggestingthat parkin mediates autophagic clearance via its E3 ubiquitin ligasefunction.

Levels of human α-Synuclein and p-Tau were measured using quantitativeELISA in subcellular fractions. A significant increase (62%, P<0.05,N=5) in the level of α-Synuclein was detected in AV-10 (FIG. 47E) andAV-20 (19%) compared to LacZ injected animals. However, co-expression ofparkin eliminated α-Synuclein from AV-10 and significantly increased itslevels in AV-20 (45%) and lysosomes (24%) compared to LacZ (FIG. 47E).Co-expression of α-Synuclein with T240R resulted in significantlyelevated (51%) levels of α-Synuclein in AV-10, and unlike wild typeparkin, failed to show any deposition in AV-20, which is enriched inautophagosomes or lysosomes. Significantly increased levels (P<0.05,N=5) of p-Tau were detected in AV-10 in animals injected withα-Synuclein (34%) or α-Synuclein+T240R (39%) compared to LacZ. However,wild type parkin expression led to a significant increase of p-Tau inAV-10 (19%) and lysosome (21%) compared to LacZ, α-Synuclein andα-Synuclein+T240R (FIG. 47F). No parkin as measured by ELISA wasdetected in subcellular fractions in these animal models, suggestingthat parkin accumulation in autophagic vesicles can take place over aprotracted time period in PD.

Functional Parkin, not Mutant T240R, Regulates Autophagic Clearance inthe Striatum of α-Synuclein Expressing Animals.

To determine the mechanisms by which parkin can mediate clearance ofautophagic vacuoles in the rat striatum, molecular markers of theautophagic pathway were examined. WB analysis showed no difference inbeclin-1 levels in animals injected with lentiviral LacZ, parkin orα-Synuclein alone (FIG. 48A). A significant increase in beclin-1 levels(54% by densitometry, N=8, P<0.05) was observed when parkin wasco-expressed with α-Synuclein, suggesting that parkin responds toα-Synuclein-induced stress. The levels of Atg7 and Atg12 were alsosignificantly increased by 41% and 33%, respectively, inparkin+α-Synuclein injected animals (FIG. 48A) compared to animalsinjected with LacZ, parkin or α-Synuclein alone. No changes in LC3-Blevels were observed between animals injected with lentiviral LacZ orparkin alone (FIG. 48B) but α-Synuclein expression significantlyincreased (51%) LC3 levels (FIG. 48B), suggesting increased amount ofautophagosomes. Co-expression of parkin and α-Synuclein decreased thelevels of LC3-B (29% by densitometry, N=8, P<0.05), suggestingdegradation of LC3-B-containing autophagic vacuoles. No changes werealso observed in HDAC6 levels (FIG. 48B) between animals injected withLacZ, parkin or α-Synuclein alone, but HDAC6 level was significantlyincreased (37%) levels (FIG. 48B) when animals were co-injected withparkin and α-Synuclein together, suggesting that parkin expressionfacilitates fusion between autophagosomes and lysosomes. No differencesin the levels of molecular markers of autophagy were observed whenmutant T240R parkin was injected either alone or with α-Synuclein. Thesedata show that parkin E3 ubiquitin ligase activity may up-regulateprotein levels of the beclin-1-dependent autophagic cascade,facilitating autophagic clearance.

The EM and WB data was supplemented with immunohistochemistry todetermine the presence of LC3-B. Staining with anti-LC3-B antibodyshowed no reactivity in the striatum of animals injected with lentiviralparkin (FIG. 48C). Lentiviral expression of α-Synuclein led to anincrease in immunoreactivity to LC3-B (FIG. 48D). Stereological countingof LC3-B positive cells revealed a significant increase (FIG. 48G. 43%,P<0.05, N=8) in striata injected with α-Synuclein. Co-injection oflentiviral parkin with α-Synuclein (FIG. 48E) resulted in disappearanceof LC3-B from the striatum. To further ascertain that functional E3ubiquitin ligase parkin mediates autophagic changes, LC3-B antibodieswere co-injected with α-Synuclein and mutant T240R parkin (FIG. 48F) instriatal sections and no elimination of LC3-B staining was observed inthese animals. Stereological counting of LC3-B stained cells in thestriatum co-injected with α-Synuclein and T240R showed a significantincrease (37%) in LC3-B reactivity compared to LacZ (FIGS. 48F&G). Tofurther determine whether wild type parkin leads to clearance ofubiquitinated proteins via autophagy we stained with anti-P62 antibody.The levels of P62 were significantly (P<0.05, N=8) increased whenα-Synuclein (41% by densitometry relative to actin) was expressedcompared to LacZ (FIG. 48F). However, parkin co-expression led tocomplete disappearance of P62 staining, suggesting autophagicdegradation of ubiquitinated proteins.

These studies show decreased parkin solubility in the striatum ofsporadic PD patients, independent of early onset disease-causingmutations. In conclusion, decreased parkin solubility can reflectdiminished parkin function, which can lead to alteration of baselineautophagy, including parkin, α-Synuclein and p-Tau clearance. Lentiviralexpression of α-Synuclein leads to p-Tau and accumulation of autophagicvacuoles. These data demonstrate an association between α-Synuclein andautophagic dysfunction in PD, and indicate a beneficial role for parkinin autophagic clearance. Thus, parkin's role in autophagic clearance canbe exploited as a therapeutic strategy in PD.

TABLE 1 Description and clinical diagnosis of human PD patients andcontrol subjects's tissues analyzed by Western blot and ELISA. FR BRC #FDX Age Sex Race PMD Area 399 Control 79 F W 24 Caudate 417 Control 80 FW 6 Caudate 487 Control 73 M W 22 Caudate 515 Control 62 M W 19 Caudate705 Control 73 M W 9 Caudate 1277 Control 80 F W 8 Caudate 2052 Control79 M W 16 Caudate 1690 PD 76 M W 18 Caudate 1731 PD 77 M W 16 Caudate2140 PD with dementia 84 F W 11 Caudate 2067 PD with dementia, 76 M W 19Caudate cerebrovas. dis (NC) 2019 PD with dementia, 83 M W 16.5 Caudatecerebrovas. dis 1989 PD with dementia, 84 M W 5 Caudate LBD neocortical2074 PD, cerebrovascular 85 F W 9 Caudate disease 1758 PD, DLB 81 M W 11Caudate 1948 PD, DLB 77 M W 5 Caudate 1796 PD, Lewy Body CHG 81 M W 8.75Caudate Limbic, porencephalic cyst 1877 PD, Lewy Body CHG 80 M W 19Caudate neocortical 1955 PD, Lewy Body CHG 84 M B 13 Caudate neocortical

TABLE 2 Description and clinical diagnosis of human PD patients andcontrol subjects stained with immonuhistohemistry. BRC # FDX CERAD BRAAKAge Sex Race PMD FX 1062 Control 58 M B 14 Hippocampus MB 1252 Control70 M W Hippocampus MB 1277 Control 0 80 F W 8 Caudate, hippocampus, MB1352 Control 78 F 14 Caudate, hippocampus, MB 1615 Control 72 M W 20Caudate 1683 Control 1 91 F W 8 Caudate 1855 Control 77 M W Caudate,hippocampus, MB 2201 Control 0 2 85 F W 27 Caudate, hippocampus, MB 2215PD with dementia, B 4 88 M W 9 Caudate, MB AD probable 2235 PD,tauopathy non- 86 F W 26 Caudate, MB AD, cerebrovas. dis (NC) 2243 PDwith dementia 0 0 68 M W 50 Caudate, MB 2253 PD, contusion 0 1 64 F W 15Caudate, MB 2267 PD with dementia, 0 1 75 M W 22 Caudate, MB neocortex2290 PD A 2 82 M W 53 Caudate, MB 2292 PD with dementia, B 4 77 M W 8Caudate, MB AD probable 2312 PD 0 0 56 M W 21 MB 2315 PD 0 2 84 M W 8.5Caudate, MB 2352 PD with dementia, 0 2 83 F W 163 Caudate, MBcerebrovas. dis (NC)

Example 3—Parkin Inactivation in Alzheimer's Disease

The role of parkin in post-mortem brain tissues from 21 Alzheimer'sdisease patients and 15 control subjects was investigated. In order todetermine the role of parkin in Aβ clearance, gene transfer animalsexpressing lentiviral Aβ₁₋₄₂ with and without parkin were generated andautophagic mechanisms were examined.

Materials and Methods

Human postmortem brain tissues. Human postmortem hippocampal andcortical regions from 21 AD patients and 15 age matched control subjectswere obtained from John's Hopkins University brain bank. The age, sex,stage of disease and postmortem dissection (PMD) are summarized for eachpatient in Table 3 and 4. The cause of death is not known. To extractthe soluble fraction of proteins, 0.5 g of frozen brain tissues werehomogenized in 1×STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mMEDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease cocktail inhibitor),centrifuged at 10,000 g for 20 min at 40 C, and the supernatants werecollected. All samples were then analyzed by ELISA (see below) orWestern blot using 30 μg of protein. To extract the insoluble fraction,the pellet was re-suspended in 4M urea solution and centrifuged at10,000 g for 15 min, and the supernatant was collected and 30 μg ofprotein was analyzed by Western blot. Western blots were quantified bydensitometry using Quantity One 4.6.3 software (Bio Rad). Densitometrywas obtained as arbitrary numbers measuring band intensity. Data wereanalyzed as mean±Standard deviation, using Two-tailed t-test (P<0.02)and ANOVA, Neumann Keuls with multiple comparisons (P<0.05) to compareAD and control groups. Insoluble parkin co-localizes with intracellularAβ.

Immunohistochemistry on slides from human patients was performed on 30μm thick paraffin embedded brain slices de-paraffinized in Xylenes 2×5minutes and sequential ethanol concentration, blocked for 1 hour in 10%horse serum and incubated overnight with primary antibodies at 4° C.After 3×10 minute washes in 1×PBS, the samples were incubated with thesecondary antibodies for 1 hr at RT, washed 3×10 minutes in 1×PBS.Aβ₁₋₄₂ was probed (1:200) with rabbit polyclonal specific anti-Aβ₁₋₄₂antibody (Zymed) that recognizes a.a.1-42, and (1:200) mouse monoclonalantibody (4G8) that recognizes a.a. 17-24 (Covance) and counterstainedwith DAPI. Parkin was immunoprobed (1:200) with mouse anti-parkin (PRK8)antibody that recognizes a.a. 399-465 (Signet Labs, Dedham, Mass.) andrabbit polyclonal (1:200) anti-parkin (AB5112) antibody that recognizesa.a. 305-622 (Millipore) and counterstained with DAPI. Because humantissues may exhibit a high level of auto-fluorescence, other experimentswere performed with the absence of either primary or secondaryantibodies to determine the specificity of immunostaining.

Stereotaxic Injection.

Lentiviral constructs were used to generate the animal models asexplained in Rebeck et al. (J. Biol. Chem. 285, 7440-7446 (2010)) andthe identity of the Aβ₁₋₄₂ species generated was confirmed by massspectroscopy. Stereotaxic surgery was performed to inject the lentiviralconstructs encoding LacZ, parkin or Aβ₁₋₄₂ into the M1 primary motorcortex of 2-month old male Sprague-Dawley rats. All animals wereanesthetized (50 mg/kg body weight) with a cocktail of Ketamine andXylazine (50:8). The stereotaxic coordinates were according to Paxinosand Watson rat brain atlas. Lentiviral stocks were injected through aMicro syringe pump controller (Micro4) using total pump (World PrecisionInstruments, Inc.) delivery of 6 μl at a rate of 0.2 μl/min. In one sideof the brain animals were injected with 1) a lentiviral-LacZ vector at2×10¹⁰ multiplicity of infection (m.o.i); 2) with 1×10¹⁰ m.o.ilentiviral-parkin and 1×10¹⁰ m.o.i lentiviral-LacZ; 3) 1×10¹⁰ m.o.ilentiviral-Aβ-Insoluble parkin co-localizes with intracellular Aβ₁₋₄₂and 1×10¹⁰ m.o.i lentiviral-LacZ; or 4) and 1×10¹⁰ m.o.ilentiviral-Aβ₁₋₄₂ and 1×10¹⁰ m.o.i lentiviral-parkin. All procedureswere approved by the Georgetown University Animal Care and Use Committee(GUACUC). A total of 84 rats were used in these studies.

Western Blot Analysis.

To extract the soluble protein fraction, brain tissues were homogenizedin 1×STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2%NP-40, 0.2% BSA, 20 mM PMSF and protease cocktail inhibitor),centrifuged at 10,000×g for 20 min at 40° C., and the supernatantscontaining the soluble fraction of proteins were collected. To extractthe insoluble fraction the pellet was re-suspended in 4M urea or 30%formic acid and adjusted to pH 7 with 1N NaOH and centrifuged at10,000×g for 20 min at 40 C, and the supernatant containing theinsoluble fraction was collected and analyzed by Western blot. Thesupernatants were analyzed by WB on SDS NuPAGE 4-12% Bis-Tris gel(Invitrogen, Inc.). Protein estimation was performed using Bio-Radprotein assay (Bio-Rad Laboratories Inc., Hercules, Calif.). Totalparkin was immunoprobed (1:1000) with PRK8 antibody as indicated [43]and phospho-parkin was probed (1:1000) with anti-Ser 378 antibodies(Pierce). Aβ₁₋₄₂ was immunoprobed with (1:600) mouse 6E10 antibody(Signet Labs, Mass.), analyzed alongside a synthetic peptide (AnaSpec,Calif., USA). Rabbit polyclonal antibodies anti-beclin (1:1000),autophagy like gene (Atg)-7 (1:1000), Atg12 (1:1000) and LC3-B (1:1000)were used to probe autophagy proteins using antibody sampler kit 4445(Cell Signaling, Inc). Histone deacetylase 6 (HDAC6) was probed (1:500)using rabbit polyclonal anti-HDAC6 (Abcam). Rabbit polyclonalanti-SQSTM1/p62 (Cell Signaling Technology) was used (1:500).Lysosomal-associated membrane protein 3 (LAMP-3) was probed (1:500) withrabbit polyclonal antibody (ProteinTech). A rabbit polyclonal (Pierce)anti-LC3 (1:1000) and rabbit polyclonal (Thermo Scientific) anti-actin(1:1000) were used.). Rabbit polyclonal (1:1000) Cyclin E (ThermoScientific), rabbit polyclonal (1:1000) tubulin (Thermo Scientific) andmouse monoclonal (1:500) anti-ubiquitin (Santa Cruz Biotechnology) wereused. Map 2 was probed (1:1000) mouse monoclonal antibody (Pierce).

Western blots were quantified by densitometry using Quantity One 4.6.3software (Bio Rad). Densitometry was obtained as arbitrary numbersmeasuring band intensity. Data were analyzed as mean±standard deviation,using ANOVA, with Neumann Keuls multiple comparison between treatmentgroups. A total number of N=8 was used in each treatment.

Immunohistochemistry was performed on 20 micron-thick 4%paraformaldehyde (PFA) fixed cortical brain sections and comparedbetween treatments. Aβ₁₋₄₂ was probed (1:200) with rabbit polyclonalspecific anti-Aβ₁₋₄₂ antibody (Zymed). Rabbit polyclonal LC3-B (1:100)was used to probe LC3-B (Cell Signaling, Inc). Thioflavin-S and nuclearDAPI staining were performed according to manufacturer's instructions(Sigma).

Stereological methods were applied by a blinded investigator usingunbiased stereology analysis (Stereologer, Systems Planning andAnalysis, Chester, Md.) to determine the total positive cell counts in20 cortical fields on at least 10 brain sections (˜400 positive cellsper animal) from each animal. These areas were selected across differentregions on either side from the point of injection, and all values wereaveraged to account for the gradient of staining across 2.5 mm radiusfrom the point of injection. An optical fractionator sampling method wasused to estimate the total number of positive cells with multi-levelsampling design. Cells were counted within the sampling frame determinedoptically by the fractionator and cells that fell within the countingframe were counted as the nuclei came into view while focusing throughthe z-axis.

Aβ, Parkin and p-Tau Enzyme-Linked Immunosorbent Assay (ELISA)—

Specific p-Tau, Aβ₁₋₄₀ and Aβ₁₋₄₂ ELISA (Invitrogen) were performedusing 50 μl (1 μl) of brain lysates detected with 50 μl human p-Tau(AT8) or Aβ primary antibody (3 h) and 100 μl anti-rabbit Insolubleparkin co-localizes with intracellular Aβ antibody (30 min) at RT.Extracts were incubated with stabilized Chromogen for 30 minutes at RTand solution was stopped and read at 450 nm, according to manufacturer'sprotocol. Parkin levels were measured using specific human ELISA(MYBioSource) was measured using human specific ELISA (Invitrogen). AllELISA were performed according to manufacturers' protocols.

Subcellular Fractionation to Isolate Autophagic Vacuoles—

Animal brains were homogenized at low speed (Cole-Palmer homogenizer,LabGen 7, 115 Vac) in 1×STEN buffer and centrifuged at 1,000 g for 10minutes to isolate the supernatant from the pellet. The pellet wasre-suspended in 1×STEN buffer and centrifuged once to increase therecovery of lysosomes. The pooled supernatants were then centrifuged at100.000 rpm for 1 hour at 40° C. to extract the pellet containingautophagic vacuoles (AVs) and lysosomes. The pellet was thenre-suspended in 10 ml (0.33 g/ml) 50% Metrizamide and 10 ml in cellulosenitrate tubes. A discontinuous Metrizamide gradient was constructed inlayers from bottom to top as follows: 6 ml of pellet suspension, 10 mlof 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide. Aftercentrifugation at 100,000 rpm for 1 hour at 40 C, the fraction floatingon the 10% layer (Lysosome) and the fractions banding at the 24%/20% (AV20) and the 20%/10% (AV10) Metrizamide inter-phases were collected by asyringe and examined.

Transmission Electron Microscopy—

Brain tissue were fixed in (1:4, v:v) 4% paraformaldehyde-picric acidsolution and 25% glutaraldehyde overnight, then washed 3× in 0.1Mcacodylate buffer and osmicated in 1% osmium tetroxide/1.5% potassiumferrocyanide for 3 h, followed by another 3× wash in distilled water.Samples were treated with 1% uranyl acetate in maleate buffer for 1 h,washed 3× in maleate buffer (pH 5.2), then exposed to a graded coldethanol series up to 100% and ending with a propylene oxide treatment.Samples are embedded in pure plastic and incubated at 60° C. for 1-2days. Blocks are sectioned on a Leica ultracut microtome at 95 nm,picked up onto 100 nm formvar-coated copper grids, and analyzed using aPhilips Technai Spirit transmission EM. All images were collected by ablind investigator.

Soluble Parkin is Decreased in Post-Mortem AD Brain Tissues.

To determine whether parkin levels are changed in other regions of ADbrain, frozen post-mortem cortical tissues from 12 AD patients and 7 agematched control subjects were examined. The clinical diagnosis andpost-mortem dissection (PMD) are summarized in Table 3. No informationwas provided about the cause of death. Western blot (WB) analysis withanti-parkin antibody (PRK8) revealed a significant decrease (52%,P<0.05) in soluble parkin level in the cortex of AD brain (FIGS. 49A &D). To ascertain that the decrease in parkin level is not due toneuronal loss, an anti-MAP-2 antibody was used as a neuronal marker andloading control (FIG. 49A). Quantitative parkin ELISA showed asignificant decrease (46%) in soluble parkin levels (FIG. 49B, P<0.05),suggesting that parkin levels may be reduced in AD. Further analysisusing two-tailed t-test (P<0.02) showed no differences within AD orcontrol samples with age, but parkin levels were significantly (P<0.05)reduced (21%) in all samples with PMD greater than 15 hours.

Potential parkin function was investigated via examination of the levelof ubiquitinated proteins and possible targets of parkin E3 ubiquitinligase activity, including tubulin and Cyclin E. The levels ofubiquitinated proteins (FIG. 49C, 1st blot) was increased in WB ofsoluble AD cortical extracts compared to control subjects, suggestinglack of degradation of ubiquitinated proteins. No significantdifferences (t-test, P<0.02) were observed within the AD group, butvariation was noticeable among control subjects, with older subjectsshowing smears of ubiquitinated proteins similar to AD (FIG. 49C, 1stblot lane 1 (case #399) and lane 3 (case #1277). Significantly increasedlevels of tubulin (2nd blot, 63%, FIG. 49D, P<0.05) and Cyclin E (3rdblot, 34%, FIG. 49D) were also observed in AD compared to controlsubjects. The insoluble protein fraction of human postmortem corticaltissues was then extracted in 4M urea and western blot was performed.Little parkin was detected in the insoluble fraction of control brains,but total parkin was significantly increased (130%, P<0.05) in ADbrains, suggesting parkin insolubility (FIGS. 49E & F). We also detectedsignificantly (P<0.05) increased levels (143%) of phosphorylated parkinat serine-378 relative to actin in AD brains but not control subjects(FIG. 49E, 2nd blot and FIG. 49F), suggesting that parkinphosphorylation may be associated with decreased solubility.

Parkin co-localizes with intraneuronal Aβ₁₋₄₂ in the hippocampus andcortex in AD. To investigate whether parkin expression is altered inhuman AD brains, a mouse monoclonal anti-parkin (PRK8) antibody thatrecognizes a.a. 399-465 and rabbit polyclonal anti-human Aβ₁₋₄₂ antibodythat recognizes a.a. 1-42 were used. Hippocampal staining showedintraneuronal Aβ₁₋₄₂ (FIG. 50A) and parkin (FIG. 50B) in nuclearDAPI-stained neurons in control human subjects, and both parkin andAβ₁₋₄₂ were detected within the same cells (FIG. 50C). The levels ofintraneuronal expression of Aβ₁₋₄₂ were increased in the hippocampus ofAD patients (FIG. 50D), without noticeable detection of amyloid plaques.Co-staining showed increased intracellular parkin levels (FIG. 50E) inAD hippocampus compared to control subjects (FIG. 50B), suggestingaccumulation of parkin in AD brains. Both intracellular parkin andAβ₁₋₄₂ were co-localized in hippocampal neurons (FIG. 50F). To ascertainthe specificity of these results in human sections alternate rabbitpolyclonal anti-parkin (AB5112) antibody that recognizes a.a. 305-622and mouse monoclonal anti-human Aβ₁₋₄₂ (4G8) antibody that recognizesa.a. 17-42 were used. Hippocampal staining showed intraneuronal Aβ₁₋₄₂(FIG. 50G) and parkin (FIG. 50H) in nuclear DAPI-stained neurons in AD,and both parkin and Aβ₁₋₄₂ were detected within the same cells (FIG.50J) without noticeable detection of amyloid plaques.

Other brain regions were examined where extracellular plaques weredetected to ascertain whether parkin co-localizes with intracellularAβ₁₋₄₂. Staining with anti-Aβ₁₋₄₂ antibody showed plaque formation inthe entorhinal cortex as well as intracellular Aβ₁₋₄₂ accumulation (FIG.51A), suggesting presence of both intracellular and extracellular Aβ₁₋₄₂in AD cortex. Parkin staining was also observed within nuclearDAPI-stained neurons in the entorhinal cortex (FIG. 51B), but parkinco-localized only with Aβ₁₋₄₂ containing neurons and not withextracellular Aβ₁₋₄₂ plaques (FIG. 51C, arrows). Further analysis of theneocortex also resulted in detection of intracellular accumulation andplaque Aβ₁₋₄₂ (FIG. 51D) in AD, as well as intracellular parkinexpression (FIG. 51E). Similarly, parkin and Aβ₁₋₄₂ were co-localized(FIG. 51F, arrows) intracellularly in AD cortex. An alternatecombination of antibodies was used (as above) and plaque formation wasdetected in the cortex as well as intracellular Aβ₁₋₄₂ accumulation(FIG. 51G), suggesting presence of both intracellular and extracellularAβ₁₋₄₂ in AD cortex. Parkin staining within nuclear DAPI-stained neuronsin AD cortex was also observed (FIG. 51H), but parkin co-localized onlywith Aβ₁₋₄₂ containing neurons and not with extracellular Aβ₁₋₄₂ plaques(FIG. 51I, arrows).

Accumulation of Parkin, Aβ and p-Tau in Autophagosomes in AD Brain.

To determine whether parkin co-staining with intracellular Aβ₁₋₄₂ isassociated with autophagic activities in AD, anti-microtubule-associatedlight chain protein 3 (LC3) antibodies were used as probles andsub-cellular fractionation was performed to measure the levels ofamyloid proteins in autophagic organelles using quantitative ELISA.Probing with anti-LC3 antibody suggested a significant increase inLC3-II compared to LC3-I (28%) levels (FIG. 52A, 1st blot & FIG. 52B),indicating possible lipidation of LC3. The amount of LC3 compared toactin was measured using an antibody specific for the LC3-B isoform. Asignificant increase (33%, P<0.05) in the level of LC3-B was detected inhuman cortical extracts from AD patients compared to control subjects(FIG. 52A, 2nd blot & FIG. 52B).

To ascertain that sub-cellular fractionation leads to isolation ofautophagic vacuoles, WB was performed with lysosomal marker usinganti-LAMP-3 antibody that showed lysosomal fraction in the top layer ofMetrazimide gradient (FIG. 52C, top blot), and anti-LC3B (2nd blot),which detected autophagosomes in both the 10% (AV-10) and 20% (AV-20)gradient fractions. The levels of Aβ and p-Tau were measured usingquantitative ELISA in these fractions. A significant increase (89%,P<0.05) in the level of Aβ₁₋₄₂ was detected in AV-10 (FIG. 52D) andAV-20 (78%), which are enriched in autophagosomes in AD compared tocontrol. Similarly, a significant increase (110%, P<0.05) in the levelof Aβ1-40 was detected in AV-10 (FIG. 52E) and AV-20 (89%) in ADcompared to control. No Aβ was detected in the lysosomal fraction. Thelevels of p-Tau were measured as another protein marker associated withAD. No p-Tau was detected in the lysosome but the levels of p-Tau (AT8)were significantly increased in AV-10 (81%) and AV-20 (140%) compared tocontrol (FIG. 52F). Because AV-20 is enriched in autophagosomes, thesedata show accumulation of un-degraded proteins in autophagosomes in AD.Surprisingly, the level of parkin was significantly increased (P<0.05)in AV-10 (64%) and AV-20 (52%) compared to control (FIG. 52G), but notin the lysosomal fraction, suggesting that accumulated parkinco-localizes with Aβ and p-Tau in autophagic compartments.

Lentiviral Aβ₁₋₄₂ induces p-Tau and amyloidogenic protein and exogenousparkin reverses these effects. Because parkin insolubility andco-localization was detected with intraneuronal Aβ₁₋₄₂ in AD brain,lentiviral gene transfer was used to co-express Aβ₁₋₄₂ and parkin in therat cortex and the effects of these proteins on autophagy wereinvestigated. It was observed that lentiviral delivery led to anincrease (50% by densitometry, N=8) of parkin expression (FIG. 53A, 1stblot) and Aβ₁₋₄₂ clearance 2 weeks post-injection of lentiviral parkintogether with Aβ₁₋₄₂ (FIG. 53A, 2nd blot). No changes in total parkinlevels were observed in brains injected with lentiviral Aβ₁₋₄₂ (FIG.53A, 1st blot), and no phospho-parkin was detected in the insoluble 4Murea or 30% formic acid fractions. To determine parkin levels,quantitative ELISA was performed for human parkin in cortical lysates.Human parkin was significantly increased in parkin (34%, N=8) orparkin+Aβ₁₋₄₂ (38%) injected animals (FIG. 53B) compared to LacZ orAβ₁₋₄₂ alone. Independent studies were then performed to determine theeffects of parkin activity on Aβ₁₋₄₂ levels in the cortex, using humanspecific Aβ₁₋₄₂ ELISA. A significant increase (FIG. 53C, 7.8-fold, N=8,P<0.05, ANOVA, Neumann Keuls with multiple comparison) in the level ofAβ₁₋₄₂ was observed 2 weeks post-injection with lentiviral Aβ₁₋₄₂ intothe cortex. Co-expression of parkin significantly decreased (6-fold)Aβ₁₋₄₂ levels, but Aβ₁₋₄₂ remained significantly higher (89%) comparedto parkin or LacZ injected animals (FIG. 53E).

The effects of parkin on amyloidogenic proteins in animals expressingAβ₁₋₄₂ were then determined. ELISA was performed and a significantincrease in rat p-Tau (AT8) was observed in the cortex at 4 weeks butnot 2 weeks post-injection (FIG. 53D). Thioflavin-S staining wasperformed to examine whether lentiviral Aβ₁₋₄₂ and p-Tau accumulationlead to formation of amyloidogenic proteins. Cortical brain sectionsshowed thioflavin-S positive staining in Aβ₁₋₄₂-expressing animals (FIG.53H) compared to lentiviral LacZ injected controls (FIG. 53E).Co-expression of parkin with Aβ₁₋₄₂ eliminated thioflavin-S positivestaining in the cortex 4 weeks post-injection (FIG. 53G). These datashow that parkin counteracts Aβ₁₋₄₂ induced amyloidogenic proteins.

Parkin Mediates Clearance of Autophagic Vacuoles Containing p-Tau andAβ₁₋₄₂.

Whether parkin expression can mediate the clearance of Aβ₁₋₄₂-inducedaccumulation of autophagic vacuoles was determined. Electron microscopyscanning of cortical sections showed no accumulation of Insoluble parkinco-localizes with intracellular Aβ vacuoles in the cytosol of lentiviralLacZ (FIG. 54A) or lentiviral parkin (FIG. 54B) injected animals.Lentiviral expression of Aβ₁₋₄₂ led to cytosolic accumulation ofautophagic vacuoles (FIG. 54C, arrows), suggesting induction ofautophagy 2-week post-injection with lentiviral Aβ₁₋₄₂. Co-expression oflentiviral parkin with lentiviral Aβ₁₋₄₂ led to formation of doublemembrane vacuoles containing debris (FIG. 54D). These data suggest thatparkin leads to autophagic clearance of lentiviral Aβ₁₋₄₂-inducedvacuoles. Sub-cellular fractionation was performed and levels of Aβ₁₋₄₂and p-Tau were measured using quantitative ELISA in these fractions. Asignificant increase (61%, P<0.05, N=5) in the level of Aβ₁₋₄₂ wasdetected in AV-10 (FIG. 54E) compared to LacZ, parkin or parkin+Aβ₁₋₄₂injected animals, indicating that Aβ₁₋₄₂ alters normal autophagy,leading to accumulation of autophagosomes. However, co-expression ofparkin led to clearance of Aβ₁₋₄₂ from AV-10 and significantly increasedAβ₁₋₄₂ levels in AV-20 (42%) and lysosomes (35%) compared to LacZ andparkin alone (FIG. 54E). Because Aβ₁₋₄₂ expression induced p-Tau at 4weeks post-injection, levels of p-Tau (AT8) were also measured. Animalsinjected with Aβ₁₋₄₂ had a significant increase (31%) in p-Tau levels inAV-10 compared to LacZ, parkin and parkin-Aβ₁₋₄₂ (FIG. 54F). However,parkin+Aβ₁₋₄₂ expression led to clearance from AV-10 and significantlyincreased p-Tau levels in AV-20 (18%) and lysosomes (20%).

Parkin Regulates Autophagosome Clearance in Aβ₁₋₄₂-Expressing Animals.

To determine the mechanisms by which parkin can mediate clearance ofautophagic vacuoles, molecular markers of the autophagic pathway,leading to autophagosomal clearance were examined. WB analysis showed nodifference in beclin levels in animals injected with lentiviral LacZ,parkin or Aβ₁₋₄₂ (FIG. 55A). However, a significant increase in beclinlevels (48% by densitometry, N=8, P<0.05) were detected when parkin wasco-expressed with Aβ₁₋₄₂, suggesting that parkin responds toAβ₁₋₄₂-induced stress. The levels of autophagy-related genes (Atgs)including Atg7 and Atg12 were also increased by 34% and 29%,respectively, in parkin+Aβ₁₋₄₂ injected animals (FIG. 55A) compared toanimals injected with LacZ, parkin or Aβ₁₋₄₂ alone. Other markers of theautophagic cascade LC3 were examined. No changes in LC3-B levels weredetected in animals injected with lentiviral LacZ or parkin alone (FIG.55B). Lentiviral Aβ₁₋₄₂ expression lead to a significant increase (32%,N=8, P<0.05) in LC3-B levels, but parkin co-expression reversed theincrease in LC3-B (FIG. 55B). A significant increase in histonedeacetylase 6 (HADC6) levels (44%) were observed in animals injectedwith lentiviral parkin+Aβ₁₋₄₂ (FIG. 55B) compared to all otherconditions. These data suggest that parkin responds to Aβ₁₋₄₂ stress viaup-regulation of molecular markers of autophagy.

The EM and WB data was supplemented with immunohistochemistry toevaluate the appearance of LC3-B staining. Staining with anti-LC3-Bantibody showed no reactivity in the cortex of animals injected withLacZ (FIG. 55C) or parkin (FIG. 55D). However, lentiviral injection ofAβ₁₋₄₂ increased LC3-B staining (FIG. 55E), in agreement with WB data.Co-injection of lentiviral parkin with Aβ₁₋₄₂ (FIG. 55F) led todisappearance of LC3-B staining. Stereological counting of LC3-Bpositive cells revealed a significant increase (FIG. 55G. 52%, P<0.05,N=8) in cortices co-injected with Aβ₁₋₄₂ compared to other treatments,indicating that parkin activity regulates autophagosome clearance inAβ₁₋₄₂ expressing animals. To further determine whether parkin leads toclearance of ubiquitinated proteins via autophagy anti-P62 antibody wasused as a probe. The levels of P62 were significantly (P<0.05, N=8)increased when Aβ₁₋₄₂ (21% by densitometry relative to actin) wasexpressed compared to LacZ (FIG. 55F). However, parkin co-expression ledto complete disappearance of P62 staining, suggesting autophagicdegradation of ubiquitinated proteins.

These studies provide the first evidence of parkin inactivity anddecreased solubility in AD. The present data show that parkin isinactivated and accumulates with Aβ₁₋₄₂ and p-Tau in autophagosomes inAD. This novel finding shows that decreased parkin E3 ubiquitin ligaseactivity can result in lack of autophagic clearance leading toaccumulation of the autophagic vacuoles observed in AD brains. The genetransfer animal studies provide evidence that lentiviral Aβ₁₋₄₂ couldinhibit autophagosome maturation similar to AD. In conclusion, thesedata demonstrate an association between parkin inactivation andco-localization with intraneuronal Aβ₁₋₄₂ with autophagic dysfunction,indicating a beneficial role for parkin in autophagic clearance. Parkininactivation could lead to decreased autophagic clearance andaccumulation of un-degraded amyloidogenic proteins in autophagosomes.Lentiviral expression of Aβ₁₋₄₂ leads to p-Tau and accumulation ofautophagic vacuoles via inhibition of autophagosome maturation and/orimpairment of transport of autophagic organelles. Parkin E3 ubiquitinligase activity enhances autophagic flux and amyloid clearance, possiblythrough increased autophagosome maturation and recognition withlysosomes. Parkin's role in autophagic clearance could be exploited as atherapeutic strategy in neurodegenerative diseases.

TABLE 3 Summary and clinical diagnoses of AD patients and controlsubjects used for biochemistry studies. FR BRC # FDX Age Sex Race PMDArea 399 Control 79 F W 24 Motor 417 Control 80 F W 6 Motor 487 Control73 M W 22 Motor 515 Control 62 M W 19 Motor 705 Control 73 M W 9 Motor1277 Control 80 F W 8 Motor 2052 Control 79 M W 16 Motor 1390 AD 75 M 12Motor 1336 AD 82 M W 10 Motor 1652 AD 85 M W 18 Motor 1657 AD 82 M W 15Motor 1697 AD/Infarcts 86 M 6 Motor 1671 AD 77 M W Motor 1801 AD 75 M 15Motor 1870 AD 85 M W 3.5 Motor 1997 AD 85 M W 5.5 Motor 2070 AD 82 M W19 Motor 2076 AD 84 F W 16 Motor 2078 AD, cerebrovas. dis (NC) 80 M 16Motor

TABLE 4 Summary and clinical diagnoses of AD patients and controlsubjects used for immuno-histochemistry studies. BRC # FDX CERAD BRAAKAge Sex Race PMD FX 1062 Control 58 M B 14 Hippocampus, MB 1252 Control70 M W Hippocampus, MB 1277 Control 0 80 F W 8 Caudate, hippocampus, MB1352 Control 78 F 14 Caudate, hippocampus, MB 1615 Control 72 M W 20Caudate 1683 Control 1 91 F W 8 Caudate 1855 Control 77 M W Caudate,hippocampus, MB 2201 Control 0 2 85 F W 27 Caudate, hippocampus, MB 1774AD C 6 87 F W 17.5 Hippocampus 1778 AD C 6 80 F W 6.5 Hippocampus, MB1782 AD 86 M W 19.5 Hippocampus, MB 1788 AD C 62 F W 36.5 Hippocampus1833 AD 79 F W 4.5 Entorhinal/Hippo 1851 AD 86 F Entorhinal/Hippo 1854AD C 6 89 M W 9.5 Hippocampus 1861 AD 85 M W 29 Hippocampus 2291 AD B 477 M W 8 Neocrotex

Example 4

Parkinson's disease is a movement disorder characterized by death ofdopaminergic (DA) substantia nigra (SN) neurons and brain accumulationof α-Synuclein. The tyrosine kinase c-Abl is activated inneurodegeneration. Lentiviral expression of α-Synuclein leads to c-Ablactivation (phosphorylation) and c-Abl expression increases α-Synucleinlevels in mouse SN, in agreement with c-Abl activation in PD brains.Lentiviral α-Synuclein induces accumulation of autophagosomes, andboosting autophagy with the c-Abl inhibitor Nilotinib increasesautophagic clearance. Nilotinib is used for adult leukemia treatment andit enters the brain within FDA approved doses, leading to autophagicdegradation of α-Synuclein and limitation of cell death, including SNneurons. Nilotinib enhances motor behavior in lentiviral PD models,increases DA levels and induces hyper-activity in transgenic A53T mice.These data show that Nilotinib can be a therapeutic strategy to degradeα-Synuclein in PD and other Synucleinopathies.

Stereotaxic Injection.

Six months old C57BL/6 mice were stereotaxically injected with 1×104m.o.i lentiviral c-Abl, α-Synuclein (or LacZ control) bilaterally intothe SN using coordinates: lateral: 1.5 mm, ventral: 4.1 mm andhorizontal: −3.64. Viral stocks were injected through a microsyringepump controller (Micro4) using total pump (World Precision Instruments,Inc.) delivery of 2 μl at a rate of 0.2 μl/min as previously described(54-56). All animal experiments will be conducted in full compliancewith the recommendations of Georgetown University Animal Care and UseCommittee (GUAUC).

Nilotinib Treatment.

Three weeks post-injection with the lentivirus, half the animals were IPtreated daily with 10 mg/Kg Nilotinib dissolved in DMSO and the otherhalf received DMSO treatments (3 μl total) for an additional 3 weeks.Half of 6-8 months old A53T transgenic mice were IP treated daily with10 mg/Kg Nilotinib and the other half DMSO.

WB Analysis.

The nigrostriatal region was isolated from α-Synuclein or c-Ablexpressing mice and compared with LacZ or total brain extracts from A53Tmice. Tissues were homogenized in 1×STEN buffer (50 mM Tris (pH 7.6),150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and proteasecocktail inhibitor), centrifuged at 10,000×g for 20 min at 40 C and thesupernatant containing the soluble protein fraction was collected. Thesupernatant was analyzed by WB on SDS NuPAGE Bis-Tris gel (Invitrogen).α-Synuclein was probed with (1:1000) mouse anti-α-Synuclein antibody (BDTransduction Laboratories, USA) or (1:1500) human antibodies(ThermoScientific). Total c-Abl was probed with (1:500) rabbitpolyclonal antibody (Thermo Fisher) and p-c-Abl (Tyr-214) with (1:500)rabbit polyclonal antibody (Millipore). β-actin was probed (1:1000) withpolyclonal antibody (Cell Signaling Technology, Beverly, Mass., USA).Autophagy antibodies, including beclin-1 (1:1000), Atg5 (1:1000), Atg12(1:1000) were used to probe according to autophagy antibody sampler kit4445 (Cell Signaling, Inc). A rabbit polyclonal (Pierce) anti-LC3(1:1000) and rabbit polyclonal (Thermo Scientific) anti-actin (1:1000)were used.). Rabbit polyclonal (1:1000) tubulin (Thermo Scientific) wasused. Map 2 was probed (1:1000) mouse monoclonal antibody (Pierce).Rabbit polyclonal anti-SQSTM1/p62 (Cell Signaling Technology) was used(1:500). WBs were quantified by densitometry using Quantity One 4.6.3software (Bio Rad).

IHC of Brain Sections.

Animals were deeply anesthetized with a mixture of Xylazine and Ketamine(1:8), washed with 1× saline for 1 min and then perfused with 4%paraformaldehyde (PFA) for 15-20 min. Brains were quickly dissected outand immediately stored in 4% PFA for 24 h at 40 C, and then transferredto 30% sucrose at 40 C for 48 h. Tyrosine Hydroxylase (TH) was probed(1:100) with rabbit polyclonal (AB152) antibody (Millipore) and humanα-Synuclein was probed (1:100) with mouse monoclonal antibodies (ThermoScientific) and DAB counterstained.

Stereological Methods.

These were applied by a blinded investigator using unbiased stereologyanalysis (Stereologer, Systems Planning and Analysis, Chester, Md.) todetermine the total positive cell counts in 20 cortical fields on atleast 10 brain sections (˜400 positive cells per animal) from eachanimal.

Cell Culture and Transfection.

Human neuroblastoma M17 cells were grown in 24 well dishes (Falcon) aspreviously described (57, 58). Transient transfection was performed with3 μg α-Synuclein, or c-Abl or beclin-1 shRNA cDNA (Open Biosystems), or3 μg LacZ cDNA for 24 hr. Cells were treated with 10 μM Nilotinib for 24hr. Cells were harvested 48 hr after transfection. Cells were harvestedone time with STEN buffer and centrifuged at 10,000×g for 20 min at 4°C., and the supernatant was collected.

Human α-Synuclein Enzyme-Linked Immunosorbent Assay (ELISA)

These were performed using 50 μl (1 μg/μl) of brain lysates (in STENbuffer) detected with 50 μl primary antibody (3 h) and 100 μlanti-rabbit secondary antibody (30 min) at RT. α-Synuclein levels weremeasured using human specific ELISA (Invitrogen) according tomanufacturers' protocols.

Caspase-3 Fluorometric Activity Assay—

To measure caspase-3 activity in the animal models, we used EnzChek®caspase-3 assay kit #1 (Invitrogen) on cortical extracts and Z-DEVD-AMCsubstrate and the absorbance was read according to manufacturer'sprotocol.

ELISA Dopamine and HVA.

Total brain or mesencephalon were collected and fresh 50 μl (1 μg/μl)brain lysates (in STEN buffer) were detected with 50 μl primary antibody(1 h) and 100 μl anti-rabbit secondary antibody (30 min) at RT accordingto manufacturer's protocols (Abnova, Cat # BOLD01090J00011) for DA and(Eagle Biosciences, Cat # HVA34-K01) for HVA.

Transmission EM.

Brain tissues are fixed in (1:4, v:v) 4% PFA-picric acid solution and25% glutaraldehyde overnight, then washed 3× in 0.1 M cacodylate bufferand osmicated in 1% osmium tetroxide/1.5% potassium ferrocyanide for 3h, followed by another 3× wash in distilled water. Samples will betreated with 1% uranyl acetate in maleate buffer for 1 h, washed 3× inmaleate buffer (pH 5.2), then exposed to a graded cold ethanol series upto 100% and ending with a propylene oxide treatment. Samples areembedded in pure plastic and incubated at 60° C. for 1-2 days. Blocksare sectioned on a Leica ultracut microtome at 95 nm, picked up onto 100nm formvar-coated copper grids and analyzed using a Philips TechnaiSpirit transmission EM. For immuno-EM studies, sections with beincubated overnight with the primary antibodies and Gold impregnated forEM analysis.

MALDI-TOF Mass Spec.

Brain extracts are freeze dried (in DMSO) and re-suspended inacetonitrile. Nilotinib quantification will carried out on a 4800MALDI-TOF—TOF Analyzer (Applied Biosystems, Calif., USA) inreflector-positive mode and then validated in MS/MS mode as previouslydescribed (54, 59). Detected fragment masses will be identified inSWISS-PROT databases using MASCOT.

Rotarod Tests.

Mice were placed on an accelerating rotarod (Columbus Instruments)equipped with individual timers for each mouse. Mice were trained tostay on the rod at a constant 5 rpm rotation for at least 2 minutes,then the speed was gradually increased by 0.2 rpm/min and the latency tofall was measured. All values were converted to % control.

c-Abl Activation is Associated with Accumulation of α-Synuclein.

To examine the relationship between c-Abl and α-Synuclein, male C57BL/6mice were stereotaxically injected with 1×10⁴ m.o.i lentiviral c-Abl, orα-Synuclein (or LacZ) bilaterally into the SN. Lentiviral α-Synucleinexpression for 6 weeks (FIG. 56A, 1st blot, 42% over LacZ level, N=9)led to an increase in total c-Abl (110%) and tyrosine 412 (T412)phosphorylated (82%) c-Abl (FIG. 56A, p<0.05, N=9) compared to actin,indicating c-Abl activation. Human post-mortem PD striatal extracts alsoshowed an increase in total (220%) and T412 (150%) c-Abl (FIGS. 56B&C,N=9) compared to control subjects (N=7, p<0.02, two-tailed t-test).Conversely, lentiviral expression of c-Abl in the mouse SN for 6 weeksled to an increase (132%) in total c-Abl (FIG. 56D, p<0.05, N=9) andT412 phosphorylation (71%) compared to actin and resulted in increasedlevels of monomeric (51%) and high molecular weight (30%) α-Synuclein,further confirming the relationship between c-Abl and α-Synucleinaccumulation.

Nilotinib is a second-generation c-Abl tyrosine kinase inhibitor (TKI)formerly known as AMN107 (35-37). Mass spectroscopy analysis revealedthat intraperotenial (IP) injection of 10 or 20 mg/kg Nilotinib intowild type mice (N=5/time point), led to detection of up to 30 ngNilotinib per mg brain tissue 3-4 hr after injection (FIG. 56E). Thelevel of Nilotinib decreased to 3.4 ng/mg 7-8 hr post-injection,indicating that Nilotinib enters the brain and is washed out within afew hours. Caspase-3 activity was then evaluated as a measure of celldeath 3 weeks post-injection with lentiviral α-Synuclein followed by 3weeks treatment with either DMSO or Nilotinib (total 6 weeks). Daily IPinjection of 10 mg/kg Nilotinib or DMSO (30 μl) for 6 weeks did notresult in any difference in caspase-3 activity in LacZ injected mice(FIG. 56F, N=32), but lentiviral α-Synuclein expression increasedcaspase-3 activity (FIG. 56F, 740%, p<0.05, N=14) and Nilotinib reversedthis increase to 140% of LacZ levels (p<0.05, N=14). Similarly, daily IPinjection of 10 mg/kg Nilotinib or DMSO (30 μl) for 3 weeks into 7-8months old transgenic model that harbors the A53T mutation ofα-Synuclein, showed an increase in caspase-3 activity (FIG. 56G, 670%, 5p<0.05, N=15) and Nilotinib reversed this increase to 101% of wild typeage-matched controls with and without Nilotinib (N=64).

c-Abl Inhibition Via Nilotinib Promotes Autophagic Degradation ofα-Synuclein.

All animals were treated daily with IP injection of 10 mg/kg Nilotinibor DMSO (A53T mice) for 3 weeks and lentiviral models were Nilotinib (orDMSO) treated 3 weeks post-injection with lentiviral α-Synuclein orLacZ. Western blot (WB) showed significant decrease in total c-Abl (78%)and T412 phosphorylated (52%) c-Abl compared to tubulin in mesencephalonneurons in lentiviral α-Synuclein mice treated daily with 10 mg/KgNilotinib compared to DMSO. Human α-Synuclein levels increased to 202ng/ml in lentiviral α-Synuclein mice treated with DMSO, and Nilotinibreversed this increase to 31 ng/ml compared to LacZ with and withoutNilotinib. Nilotinib treatment resulted in decreased levels of monomeric(42%) and high molecular weight α-Synuclein compared to actin level. Anincrease in several molecular markers of autophagy including beclin-1(62%), Atg-5 (43%) and Atg-12 (58%) were observed compared to actin.Further analysis of autophagic markers showed significant decreases inP62 (69%) and LC3-II compared to both LC3-I (39%) and MAP-2 (41%) withNilotinib treatment, suggesting autophagic clearance of α-Synuclein.Similarly, daily IP injection of Nilotinib for 3 weeks into 7-8 monthsA53T mice, which do not express α-Synuclein in the SN, showedsignificant decrease in total c-Abl (64%) and T412 phosphorylated (70%)c-Abl compared to MAP-2 in total brain extracts compared to DMSO treatedmice. An increase in the level of total (109%) and T412 (76%) c-Abl wereobserved in A53T mice treated with DMSO compared to age-matchedcontrols. A significant increase in LC3-II level was observed inA53T+DMSO mice compared to control and LC3-II completely disappeared inA53T mice treated with Nilotinib, suggesting autophagic clearance. Humanα-Synuclein levels were increased to 785 ng/ml in A53T mice treated withDMSO, and Nilotinib reversed this increase to 467 ng/ml compared tocontrol. Nilotinib treatment resulted in decreased levels of monomeric(41%) and high molecular weight human α-Synuclein compared to actinlevel. No differences in beclin-1 and Atg5 levels were observed betweenA53T+DMSO mice and wild type control, but an increase in Atg12 (24%) wasnoted compared to actin. However, Nilotinib increased beclin-1 (69%) andAtg-5 (29%) compared to DMSO treatment in A53T mice.

To further determine whether autophagy mediates α-Synuclein clearance,human M17 neuroblastoma cells were transfected with 3 μg lacZ,α-Synuclein or shRNA beclin-1 for 24 hr and then treated with 10 μMNilotinib for additional 24 hr. An increase in α-Synuclein (264 ng/ml)was observed in α-Synuclein transfected cells compared to LacZ (FIG.57H, p<0.05, N=12) treated with 1 μl DMSO. Nilotinib reversedα-Synuclein to 35 ng/ml (p<0.05) but blocking beclin-1 expression withshRNA attenuated Nilotinib clearance of α-Synuclein (150 ng/ml) comparedto DMSO (251 ng/ml), suggesting autophagic involvement in α-Synucleinclearance.

Nilotinib clears α-Synuclein and protects SN Tyrosine hydroxylase (TH)neurons. Immunohistochemical staining of 20 μm thick brain sectionsshowed human α-Synuclein expression in mice injected with lentiviralα-Synuclein into the SN and treated with DMSO (FIG. 57B) compared toLacZ+Nilotinib (or DMSO) mice (FIG. 57A, N=12) and Nilotinib led to 84%(by stereology) decrease of human α-Synuclein (FIG. 57C, p<0.05, N=12)in SN neurons. A significant decrease in TH+ neurons (89% by stereology)was observed in lentiviral α-Synuclein+DMSO (FIGS. 57E&H) compared toLacZ+Nilotinib (FIGS. 57D&G) mice, and Nilotinib treatment ofα-Synuclein expressing mice reversed TH+neuron loss back to 82% (FIGS.57F&I, by stereology) of LacZ level (p<0.05, N=12). Stereologicalcounting showed a similar decrease (72%) of Nissl counter-stainedTH+cells in α-Synuclein+DMSO (FIG. 57K) compared to LacZ (FIG. 57J) and64% of α-Synuclein+Nilotinib (FIG. 57L, p<0.05, N=12), suggesting thatα-Synuclein causes cell death and not down-regulation of TH.Transmission electron microscopy of SN neurons showed accumulation ofcytosolic debris (FIG. 58A) and autophagic vacuoles (AV) in Lentiviralα-Synuclein expressing mice with DMSO treatment. Accumulation ofcytosolic AVs containing debris was observed in these animals (FIGS.58C&E), suggesting autophagosome accumulation, consistent with increasedLC3-II by WB. Nilotinib treatment led to appearance of larger AVs thatseemed to be derived from fusion of multiple autophagic compartments(FIGS. 58B, D &F).

Nilotinib Attenuates α-Synuclein Levels in A53T Mice.

Immunohistochemical staining of 20 μm 7 thick brain sections showedabundant expression of human α-Synuclein in the striatum of 6-8 monthsold transgenic A53T mice treated with DMSO (FIG. 59A), brainstem (FIG.59B), cortex (FIG. 59C) and Hippocampus (FIG. 59D). No α-Synucleinstaining was detected in SN of A53T mice. Daily IP injection ofNilotinib for 3 weeks led to striatal decrease (72%) of humanα-Synuclein (FIG. 59E), completely eliminated α-Synuclein from brainstem(FIG. 59F), and reduced cortical (FIG. 59G, 71%) and hippocampal (FIG.59H, 81%) α-Synuclein (p<0.05, N=7) in transgenic A53T mice.

Nilotinib Increases DA Level and Improves Motor Performance.

To evaluate α-Synuclein and Nilotinib effects on DA metabolism, DA andits metabolite Homovanilic acid (HVA) were measured using ELISA. Asignificant decrease (p<0.05, N=8) in DA (62%) and HVA (36%) wereobserved in brain mesencephalon extracts of lentiviral α-Synuclein+DMSOcompared to LacZ mice with and without Nilotinib. However, Nilotinibinjection significantly (P<0.05, N=8) reversed DA and HVA loss back tocontrol lacZ levels Lentiviral α-Synuclein expression in SN decreasedrotarod motor performance to 39% of LacZ controls with and withoutNilotinib, but Nilotinib treatment of α-Synuclein mice reversed motorperformance to 86% of LacZ level, suggesting that reversal of DA levelsleads to improved motor performance. No loss of DA or HVA were observedin transgenic A53T mice treated with DMSO compared to age-matchedcontrol with and without Nilotinib, but Nilotinib dramatically increasedboth DA (174%) and HVA (50%) levels in A53T mice. No noticeabledifferences of rotarod performance were observed between 6-8 months oldA53T mice treated with DMSO and wild type controls. However, Nilotinibincreased rotarod motor performance (45%) above control levels,suggesting hyperactivity in A53T mice.

Example 5

The tyrosine kinase c-Abl is activated in neurodegenerative disorders,including Alzheimer's disease (AD). Nilotinib is a c-Abl inhibitorapproved by the U.S. Food and Drug Administration (FDA) for treatment ofadult leukemia. These studies show that Nilotinib-mediated parkinactivation stimulated the autophagic clearance pathway, leading toamyloid degradation and cognitive improvement in a parkin-dependentmanner. Nilotinib failed to clear autophagic vacuoles and amyloidproteins in parkin−/− mice, despite an increase in beclin-1 levels,whereas beclin-1 knockdown attenuated Aβ clearance, underscoring anindispensable role for endogenous parkin in autophagy. These data showedthat Nilotinib-mediated c-Abl inhibition is a therapeutic strategy torescue cells from intraneuronal amyloid toxicity and prevent both plaquedeposition and progression from mild cognitive impairment to AD.

Human Postmortem Brain Tissues.

Human postmortem samples were obtained from John's Hopkins Universitybrain bank. Patients' description and sample preparation are summarizedin Example 1. Data were analyzed as mean±Standard deviation, usingTwo-tailed t-test (P<0.05).

Stereotaxic Injection.

Lentiviral constructs encoding LacZ, or Aβ₁₋₄₂ were stereotaxicallyinjected 1×106 multiplicity of infection (m.o.i) bilaterally into theCA1 hippocampus of 1 year old C57BL/6 or parkin−/−. A Total of 6Wlentiviral stocks were delivered at a rate of 0.2 μl/min and. Allprocedures were approved by the Georgetown University Animal Care andUse Committee (GUACUC).

Nilotinib Treatment.

Nilotinib was dissolved in DMSO and a total volume of 30 μl wereintra-peroteneally (IP) injected once a day for 3 weeks. Half theanimals received DMSO and the other half received Nilotinib in DMSO.

Western Blot Analysis.

Brain tissues were homogenized in 1×STEN buffer, centrifuged at 10,000×gfor 20 min at 40 C, and the supernatants containing the soluble fractionof proteins were collected. The pellet was re-suspended in either 4Murea or 30% formic acid and adjusted to pH 7 with 1N NaOH andcentrifuged at 10,000×g for 20 min at 4° C., and the supernatantcontaining the insoluble fraction was collected. Total parkin wasimmunoprobed (1:1000) with PRK8 antibody. Rabbit polyclonal antibodiesanti-beclin-1 (1:1000), autophagy like gene (Atg)-5 (1:1000), Atg12(1:1000) and LC3-B (1:1000) were used to probe autophagy proteins usingantibody sampler kit 4445 (Cell Signaling, Inc). A rabbit polyclonal(Pierce) anti-LC3 (1:1000) and rabbit polyclonal (Thermo Scientific)anti-actin (1:1000) were used. Rabbit polyclonal (1:1000) tubulin(Thermo Scientific) and mouse monoclonal (1:500) anti-ubiquitin (SantaCruz Biotechnology) were used. Map 2 was probed (1:1000) mousemonoclonal antibody (Pierce).

Immunohistochemistry.

Immunohistochemistry was performed on 20 micron-thick 4%paraformaldehyde (PFA) fixed cortical brain sections. Aβ1-42 was probed(1:200) with rabbit polyclonal specific anti-Aβ1-42 antibody (Zymed)that recognizes a.a.1-42, and (1:200) mouse monoclonal antibody (4G8)that recognizes amino acid 17-24 (Covance) and counterstained with DAPI.Parkin was immunoprobed (1:200) with mouse anti-parkin (PRK8) antibodythat recognizes amino acid 399-465 (Signet Labs, Dedham, Mass.) andrabbit polyclonal (1:200) anti-parkin (AB5112) antibody that recognizesamino acid 305-622 (Millipore) and counterstained with DAPI. Mousemonoclonal (6E10) antibody (1:100) with DAB were used (Covance) andthioflavin-S was performed according to manufacturer's instructions(Sigma).

Stereological Methods.

Stereological methods were applied by a blinded investigator usingunbiased stereology analysis (Stereologer, Systems Planning andAnalysis, Chester, Md.) as described in (20,36).

ELISA.

Aβ and p-Tau enzyme-linked immunosorbent assay (ELISA) using specificp-Tau, Aβ1-40 and Aβ₁₋₄₂ ELISA and caspase-3 activity were performedaccording to manufacturer's protocol.

Transmission Electron Microscopy.

Brain tissue were fixed in (1:4, v:v) 4% paraformaldehyde-picric acidsolution and 25% glutaraldehyde and analyzed by a blind investigator asdescribed in (20,36).

Cell Culture and Transfection.

Human neuroblastoma M17 or rat B35 cells were grown in 24 well dishes(Falcon). Transient transfection was performed with 3 μg Aβ₁₋₄₂ cDNA, or3 μg LacZ cDNA for 24 hr. Cells were treated with 10 μM Nilotinib for 24hr. Cells were harvested 48 hr after transfection. Cells were harvestedone time with STEN buffer and centrifuged at 10,000×g for 20 min at 4°C., and the supernatant was collected.

Parkin ELISA.

ELISA was performed on brain soluble brain lysates (in STEN buffer) orinsoluble brain lysates (4M urea) using mouse specific parkin kit(MYBioSource) in 50 μl (1 μg/μl) of brain lysates detected with 50 μlprimary parkin antibody (3 h) and 100 μl anti-rabbit antibody (30 min)at RT. Extracts were incubated with stabilized Chromogen for 30 minutesat RT and solution was stopped and read at 450 nm, according tomanufacturer's protocol.

Parkin E3 Ubiquitin Ligase Activity.

To determine the activity of parkin E3 ligase activity, E3LITECustomizable Ubiquitin Ligase Kit (Life Sensors, UC #101), whichmeasures the mechanisms of E1-E2-E3 activity in the presence ofdifferent ubiquitin chains was used. To measure parkin activity in thepresence or absence of substrates, parkin was immunoprecipitated (1:100)with PRK8 antibodies. UbcH7 was used as an E2 that provides maximumactivity with parkin E3 ligase and added E1 and E2 in the presence ofrecombinant ubiquitin, including wild type or no lysine mutant (K0), orK48 or K63 to determine the lysine-linked type of ubiquitin. E3 wasadded as IP parkin to an ELISA microplate that captures poly-ubiquitinchains formed in the E3-dependent reaction, which was initiated with ATPat room temperature for 60 minutes. Controls included, E1-E2-E3 and apoly-ubiquitin chain control in addition to E1, E2 and Aβ₁₋₄₂ withoutparkin and assay buffer for background reading. The plates were washed 3times and incubated with streptavidin-HRP for 5 minutes and were read ona chemiluminecense plate reader.

20S Proteasome Activity Assay.

Brain extracts 100 μg were incubated with 250 μM of the fluorescent 20Sproteasome specific substrate Succinyl-LLVY-AMC at 37° C. for 2 h. Themedium was discarded and proteasome activity was measured in tissuehomogenates.

Morris Water Maze.

All animals were pre-trained (trials) to swim for 90 seconds in a watermaze containing a platform submerged in water (invisible) for 4consecutive days once a day. The pretraining trials “teach” the swimminganimals that to “escape”, they must find the hidden platform, and stayon it. The water maze “test” was performed on day 5, (40), when theplatform was removed and mice have to swim and find it, thus assessingacquisition and retention. All parameters, including distance travelledto reach platform, speed to get to the platform, latency or time spenton platform, and platform entry were digitally recorded on a computerand analyzed by a blind investigator.

Novel Object Recognition (NOR).

Mice were placed individually in a 22×32×30 cm testing chamber for a 5min habituation interval and returned to their home cages. Thirtyminutes later mice were placed in the testing chamber for 10 min withtwo identical objects (acquisition session), then returned to their homecages and 90 later placed back in the testing chamber in the presencewith one of the original objects and one novel object of the same sizebut of a different color and shape (recognition session). Sessions werevideo recorded. Time spent exploring the objects were scored by blindobserver. The recognition index was calculated as (time exploring one ofthe objects/time exploring both objects)×100 for acquisition session,and (time exploring new object/time exploring both familiar and novelobjects)×100 for the recognition session. Statistical calculations toestimate differences between sessions were performed with a pairwiset-test.

Nilotinib Activates Parkin and Induces Autophagic Clearance in aBeclin-1-Dependent Manner.

To test Nilotinib effects on autophagic mechanisms, human M17 or rat B35neuroblastoma cells were transfected with 3 μg of human cDNA Aβ₁₋₄₂ (orLacZ) for 24 hr, and then treated these cells with severalconcentrations (1 nM, 100 nM, 1 μM and 10 μM) of Nilotinib for 24 hr. Nocell death (by MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) was detected in LacZ cells treated with DMSO or Nilotinib (FIG.60A). Cells expressing Aβ₁₋₄₂ had a significant level of cell death(62%, p<0.05, N=12) that was reversed to 83% of control level byNilotinib (FIG. 60A, N=12), suggesting protective effects for Nilotinibagainst Aβ₁₋₄₂ toxicity. Parkin levels were measured via ELISA usingparkin−/− brain extracts as a specificity control (FIG. 66A). Anon-significant increase (17%) in parkin was observed with 10 μMNilotinib but a significant increase (24%, FIG. 78A, N=12, p<0.05) wasreached in extracts treated with Aβ₁₋₄₂ +Nilotinib, suggesting thatparkin increases in response to Aβ₁₋₄₂ stress. To determine whetherparkin increase is associated with proteasome activity, theChymotrypsin-like assay was used with the 20S proteasome inhibitorlactacystin as a specificity control (FIG. 60B). Nilotinib did notaffect proteasome activity in LacZ cells (FIG. 60B). Proteasome activitywas increased (43%, p<0.05, N=12) in Aβ₁₋₄₂ cells, an effect that wasreversed by treatment with Nilotinib. Soluble (STEN buffer), insoluble(30% formic acid) and cell culture medium levels of Aβ₁₋₄₂ were measuredafter Nilotinib treatment. The level of secreted Aβ₁₋₄₂ was (6-fold)higher than LacZ cells; and Nilotinib decreased this by 24% (FIG. 60C,p<0.05, N=12). Nilotinib completely reversed the 2-fold increase insoluble and 3.5-fold increase in insoluble Aβ₁₋₄₂.

Lentiviral Parkin Injected into AD Mice Increases Beclin-1 Levels andAutophagic Clearance of Aβ.

Blocking beclin-1 expression using shRNA (FIG. 60D, top blot) resultedin a significant increase (FIG. 60D, 28%, N=12) in Nilotinib-inducedparkin. Aβ₁₋₄₂ levels were unaffected in the media with Nilotinibtreatment compared to Aβ₁₋₄₂ expressing cells, and were significantlyhigher than Aβ₁₋₄₂ +Nilotinib (FIG. 60C). Soluble and insoluble Aβ₁₋₄₂were partially (42% and 21%, respectively) decreased compared to Aβ₁₋₄₂transfected cells, but remained 2-fold higher compared to Aβ₁₋₄₂+Nilotinib (FIG. 60C, p<0.05, N=12), indicating that beclin-1 isrequired for complete Aβ₁₋₄₂ clearance. Secreted Aβ₁₋₄₂ (media) may haveaccumulated in the first 24 hr after transfection, prior to Nilotinibtreatment. To verify whether autophagy is involved in Aβ₁₋₄₂ clearance,LC3 (Light Chain Protein-3) levels (FIG. 60D) were examined and LC3-IIwith expression of Aβ₁₋₄₂ alone, or with shRNA beclin-1+Nilotinib (N=12)was detected, indicating autophagosome formation, but LC3-II completelydisappeared with Nilotinib, suggesting autophagic clearance (FIG. 60D).To determine Nilotinib effects on parkin function, parkin (E3) wasimmumoprecipitated and E1, E2 (UbcH7) was added and either wild typeubiquitin containing all seven lysines or the no-lysine mutant ubiquitin(K0). Nilotinib (24 hr) significantly increased parkin selfpoly-ubiquitination compared to DMSO and specificity controls (FIG. 60E,170%, N=5, P<0.05), including recombinant E1-E2-E3 (positive) or K0(negative) (FIG. 60E), suggesting that Nilotinib increases parkin E3ubiquitin ligase activity.

Nilotinib Crosses the Blood Brain Barrier.

To determine whether Nilotinib crosses the blood brain barrier 2-monthold C57BL/6 mice were intraperoteneally (IP) injected with 10 mg/kg, 20mg/kg or 50 mg/kg Nilotinib (304, in DMSO) and the animals weresacrificed 4-6 hr after injection. Mass spectroscopy analysis of totalbrain lysates showed up to 30 ng/ml Nilotinib in the brain with 10mg/kg. Nilotinib treatment (N=35) daily for 9 consecutive dayssignificantly decreased (44%) total c-Abl levels and T412 (50%),suggesting c-Abl inhibition. This treatment with 10 mg/kg Nilotinibdecreased pan-tyrosine phosphorylation, increased (29%, p<0.05, N=35)parkin and decreased ubiquitinated protein smear.

To determine Nilotinib effects on neuronal death, 1×10⁶ m.o.i lentiviralAβ₁₋₄₂ was stereotaxically injected bilaterally into the hippocampus of1 year old C57BL/6 (wild type) or parkin−/− mice and 3 weeks later 10mg/kg as injected once a day for 3 additional weeks. No differences incaspase-3 activation were observed between DMSO and Nilotinib treatedwild type mice (FIG. 60F, N=64); however, a significant increase (165%)in caspase-3 activation was observed in lentiviral Aβ₁₋₄₂ age-matchedmice (FIG. 60F, p<0.05, N=35), while Nilotinib significantly reversed(45% above control) the effects of Aβ₁₋₄₂. Similarly, no differences incaspase-3 activation were observed between DMSO and Nilotinib treatedparkin−/− mice (FIG. 60F, N=16), but a significant increase was observedin lentiviral Aβ₁₋₄₂ mice with (165%) or without (180%) Nilotinib(P<0.05, N=19), suggesting that Nilotinib depends on parkin to protectagainst Aβ₁₋₄₂.

Nilotinib Clearance of Brain Amyloid is Associated with ParkinActivation.

To determine whether Nilotinib affects Aβ level in vivo, 8-12 months oldAD transgenic mice which express neuronally derived human APP gene, 770isoform, containing the Swedish K670N/M671L, Dutch E693Q and Iowa D694Nmutations (Tg-APP) under the control of the mouse thymus cell antigen 1,theta, Thy1, promoter were treated (10 mg/kg IP injection) for 3 weeks.These mice expressed significantly higher levels of soluble (156 ng/ml)and insoluble (173 ng/ml) Aβ₁₋₄₂ compared to 1-year old control with andwithout Nilotinib (FIG. 61A, p<0.05, N=9) while Nilotinib greatlyreduced soluble Aβ₁₋₄₂ (35 ng/ml, which remained significantly higherthan control) and reversed the increase in insoluble Aβ₁₋₄₂. Significantincreases in soluble (281 ng/ml) and insoluble (250 ng/ml) Aβ1-40 werealso detected in Tg-APP mice compared to 1-year old control (FIG. 61B,p<0.05, N=9), and were reversed by Nilotinib. p-Tau was also increasedat ser 396 (109 ng/ml) and AT8 (288 ng/ml) compared to 1-year oldcontrol (FIG. 61C, p<0.05, N=9). Nilotinib significantly reduced but didnot completely reverse these increases.

Nilotinib Abrogates Alteration of Parkin Solubility in AD Mice.

To determine whether parkin level is affected in AD models, the level ofparkin was measured in Tg-APP mice in both the soluble (STEN) andinsoluble (4M urea) fractions. Brain lysates from parkin−/− mice wereused as specificity controls. No changes in soluble or insoluble parkinwere detected in control mice with and without Nilotinib (FIG. 61D,N=9). However, Nilotinib significantly increased the level of solubleparkin from 64 ng/ml in Tg-APP+DMSO to 119 ng/ml (FIG. 61D, N=11,p<0.05) while it significantly decreased insoluble parkin level from 54ng/ml to 31 ng/ml in Nilotinib treated mice (FIG. 61D, p<0.05, N=11).These data suggest increased levels of insoluble parkin in Tg-APP.Western blot revealed increased levels of total (51%) and T412 c-Abl(64%) in Tg-APP compared to control (FIG. 61E, p<0.05, N=11), whileNilotinib again reversed these increases (FIG. 61E, p<0.05, N=11). c-Ablinhibition with Nilotinib reduced the level of CTFs (44%, p<0.05, N=11)relative to MAP-2.

c-Abl Activation is Associated with Decreased Parkin Level inPost-Mortem AD Cortex.

Whether c-Abl is altered in human post-mortem AD cortex was examined(N=12 AD and 7 control). Significantly increased levels (90%) of total(FIG. 61F) and T412 (184%) c-Abl were detected in AD brains (FIG. 61F).The ratio of p-cAbl over total c-Abl (FIG. 61G) was also increased(102%). In contrast, parkin was decreased (70%) in AD cortex (FIGS.61F&G) compared to actin. Parkin insolubility may be associated withloss of E3 ligase function, so it was determined whether endogenousparkin can mediate Aβ₁₋₄₂ clearance. Significant increases (p<0.05,N=12) in soluble (180 ng/ml) and insoluble (209 ng/ml) Aβ₁₋₄₂ wereobserved in wild type lentiviral Aβ₁₋₄₂ mice (FIG. 61H), but Nilotinibcompletely reversed Aβ₁₋₄₂ back to control level. Lentiviral Aβ₁₋₄₂ inparkin−/− mice (FIG. 61H) significantly increased soluble (241 ng/ml)and insoluble (246 ng/ml) Aβ₁₋₄₂ compared to lentiviral Aβ₁₋₄₂ in wildtype mice (N=12). Interestingly, Nilotinib failed to clear soluble (297ng/ml) and insoluble (274 mg/ml) Aβ₁₋₄₂ in parkin−/− mice, suggestingthat endogenous parkin is required for Aβ₁₋₄₂ clearance. Similarly,Nilotinib decreased p-Tau ser 396 (FIG. 61I) in wild type mice (68ng/ml) compared to Aβ₁₋₄₂ expression (124 ng/ml) while p-Tau wasincreased (264 ng/ml) in parkin−/− mice and Nilotinib did not lowerp-Tau (189 ng/ml) level (FIG. 61I, p<0.05, N=11).

Nilotinib Promotes Autophagic Clearance of Amyloid in a Parkin-DependentManner.

Western blot (WB) of total brain lysates in 1 year old wild type miceinjected with lentiviral Aβ₁₋₄₂ showed a significant decrease in total(55%) and T412 (45%) c-Abl following daily treatment with 10 mg/kgNilotinib for 3 weeks (FIG. 62A, p<0.05, N=9). A significant decrease(38%) in LC3-B and disappearance of LC3-II (which indicatesautophagosome accumulation) were observed in Nilotinib compared to DMSOtreated mice (FIG. 62A, p<0.05, N=9). No changes in the neuronal markerMAP-2 (loading control) were detected. A significant increase in parkinlevel (62%) was associated with a similar increase in beclin-1 (53%) andother molecular markers of autophagy, including Atg5 (34%) and Atg12(41%) relative to tubulin (FIG. 62B, p<0.05, N=9), consistent with thehypothesis that c-Abl inhibition may mediate autophagic clearance viaincreased parkin activity. Nilotinib treatment of Aβ₁₋₄₂ mice alsoincreased parkin, decreased autophagic markers LC3-B and LC3-II (FIG.62C, p<0.05, N=12), and increased beclin-1 (53%) and Atg5 (62%) comparedto DMSO (p<0.05). Total Tau was unaffected in Tg-APP mice between DMSOand Nilotinib groups (FIG. 62D, N=12). A significant decrease in AT8(71%), AT180 (34%) and Ser 396 (64%) with no change in p-Tau Ser 262compared to actin (FIG. 62D, P<0.05) were observed in Nilotinib treatedAβ₁₋₄₂ mice.

Nilotinib effects also were examined in lentiviral Aβ₁₋₄₂ treatedparkin−/− and wild type mice (FIGS. 62E&F). Interestingly, parkin−/−mice had significantly higher levels of autophagic markers, includingbeclin-1 (FIG. 62E, 120%, N=9) compared to control. Nilotinib did notclear LC3-II in parkin−/− mice and no difference was observed in LC3-Abetween parkin−/− and control mice (FIG. 62E). Significant increases inAtg12 (FIG. 62F, 64%) and Atg5 (FIG. 62F, 74%) were observed inparkin−/− compared to control and the levels of these markers also werenot changed in response to Nilotinib. These data indicate that despitethe compensatory increase in autophagic markers, Nilotinib cannot clearautophagosomes in parkin−/− mice, further suggesting that parkin isessential for autophagosome maturation.

Nilotinib Increases Parkin Level and Decreases Plaque Load in Tg-APPMice.

Staining of 20 μm brain sections shows plaque formation within variousbrain regions in Tg-APP mice treated with DMSO (FIG. 63A-D representingdifferent animals), though plaque staining disappeared in the Nilotinibgroup after 3-week treatment (FIG. 63E-H). These results were confirmedby thioflavin-S staining (FIG. 67). Higher magnification showsendogenous parkin associated with Tg-APP (FIG. 63I) and plaquedeposition (FIGS. 63I &K) in the hippocampus. Nilotinib increasesendogenous parkin (FIG. 63L) and results in plaque disappearance (FIGS.63M&N). Using different parkin antibodies to show parkin (FIG. 63O) andplaque (FIGS. 63P&Q), Nilotinib increased parkin levels (FIG. 63R) anddissolved plaques (FIGS. 63S&T). To determine whether parkin targetsintracellular Aβ to decrease extracellular plaque load, lentiviralinjection was used to show intracellular Aβ₁₋₄₂ within the hippocampus(FIG. 63U, inset higher magnification) and Nilotinib clearance ofintracellular Aβ₁₋₄₂ (FIG. 63V, inset is higher magnification).Lentiviral injection into the hippocampus led to intracellular Aβ₁₋₄₂expression throughout the cortex (FIG. 63W, inset higher magnification)and, again, Nilotinib eliminated Aβ₁₋₄₂ accumulation (FIG. 63X, insethigher magnification). Lower magnification images show formation ofplaques in Aβ₁₋₄₂ expressing mice 6 weeks post-injection (FIGS. 64A-C).Nilotinib (daily for 3 weeks) eliminates plaque formation in Aβ₁₋₄₂ wildtype mice (FIGS. 64D-F). Aβ₁₋₄₂ expression in parkin−/− mice showed moreplaque formation (FIGS. 64G-I) and Nilotinib did not reduce plaque loadin these mice (FIGS. 64J-L). Quantification of plaque size using Image Jto delineate boundaries around individual plaques (N=15-25 plaques, 2plaques per animal) (FIGS. 68A-D) showed an average plaque size around48 μm (FIGS. 68A&I, N=12) in Aβ₁₋₄₂ wild type mice, while Nilotinibreduced plaque size to 5 μm (FIGS. 68B&I, p<0.05,). In contrast, plaquesize was larger in parkin−/− mice (FIGS. 68C&I, 85 μm, N=6), andNilotinib did not reduce plaque size (FIGS. 68D&I, 79 μm). Stereologicalcounting of Aβ-42 positive cells showed significantly reduced (N=5200cells) staining in Nilotinib treated (FIGS. 68F&J) compared to DMSOtreated Aβ₁₋₄₂ expressing wild type mice (FIGS. 68E&J, p<0.05). However,parkin−/− mice had significantly fewer Aβ₁₋₄₂ positive cells (FIGS.68G&J, N=14566) and Nilotinib did not alter intracellular staining(FIGS. 68H&J, N=13250), raising the possibility that endogenous parkincan modify intracellular Aβ₁₋₄₂, leading to intraneuronal degradation,thus limiting its secretion.

Parkin Mediates K63-Linked Ubiquitination of Aβ₁₋₄₂.

To determine whether parkin mediates any specific poly-ubiquitinlinkages of Aβ₁₋₄₂ that would facilitate its degradation, parkin wasimmunoprecipitated and synthetic Aβ₁₋₄₂ was used as a substrate. Acocktail of recombinant E1-E2-E3 and poly-ubiquitin chains were used aspositive controls (FIG. 68K). No activity was detected with lysine nullubiquitin (K0), and parkin activity was not significantly altered withK48 ubiquitin mutant. However, poly-ubiquitin signals were significantlyincreased (89%) in the presence of Aβ₁₋₄₂ compared to parkin alone (FIG.68K, p<0.05, N=6) with K63 ubiquitin, suggesting that parkin promotesK63-linked poly-ubiquitination of Aβ₁₋₄₂. Poly-ubiquitin signals werealso significantly higher with wild type ubiquitin in the presence ofAβ₁₋₄₂ (43%).

Impairment of Autophagic Clearance in the Absence of Parkin.

Transmission electron microscopy revealed (N=6 animals per treatment)autophagic defects in lentiviral Aβ₁₋₄₂ expressing mice, manifested inhippocampal appearance of dystrophic neurons (FIG. 64M), accumulation ofundigested vacuoles in the cortex (FIG. 64N) and enlargement ofhippocampal lysosomes (FIG. 64O), suggesting deficits in proteolyticdegradation. Nilotinib reversed these effects in the hippocampus (FIGS.64P&R), where no dystrophic neurons or lysosomal enlargement weredetected, and contributed to cortical clearance of vacuoles (FIG. 64Q).In contrast, Nilotinib failed to eliminate dystrophic neurons in thehippocampus of parkin−/− mice (FIGS. 64S&V), and was unable to clearvacuoles in cortex and hippocampus (FIGS. 64T-X).

Nilotinib Improves Cognitive Performance in a Parkin-Dependent Manner.

The Morris water maze test was performed after 4 days of training trialsin which the platform was placed in the SE corner and mice wereinitially placed in the NW corner of the pool. Aβ₁₋₄₂-injected (+DMSO)mice remained longer (24%) in the NW quadrant compared to control(LacZ+Nilo) (FIG. 65A, N=14), while Nilotinib reversed time (in seconds)spent in NW back to the level observed in control mice. Aβ₁₋₄₂ parkin−/−mice (N=7) with and without Nilotinib remained significantly more in theNW quadrant (FIG. 65A). Aβ₁₋₄₂ expressing wild type and parkin−/− spentsignificantly less time in SE (FIG. 65A, 47%, p<0.05) compared tocontrol, but Nilotinib significantly improved time spent in SE in wildtype but not parkin−/− compared to control (26%) and DMSO (61%). A heatmap for each group showed that controls learned quickly to find (SE)platform area, and Aβ₁₋₄₂ (DMSO) animals spent more time roaming, whileNilotinib improved platform search. In contrast, parkin−/−±Nilotinibwandered aimlessly in the maze. Aβ₁₋₄₂ animals entered the SE (platformentry, clear bars) less (FIG. 77B, 37%) than control, but Nilotinibreversed the number of entries back to control, while parkin−/− enteredsignificantly less (34%, P<0.05, N=7), suggesting that Nilotinibenhanced memory in a parkin-dependent manner. However, the distancetravelled (FIG. 77B, back bars) by Aβ₁₋₄₂ parkin−/−±Nilotinib wassignificantly decreased (80% and 75%, respectively) compared to controland wild type (P<0.05).

These experiments were repeated in 1 year old Tg-APP mice andage-matched control (C57BL/6). Tg-APP (+DMSO) mice remained less (24%)in NW (FIG. 77C, 28%, N=12) and spent significantly less time in SE(FIG. 77C, 28%, p<0.05). Nilotinib treatment (10 mg/kg daily for 3weeks) significantly reversed time spent in SE back to control level. Aheat map for each group shows that Tg-APP did better in finding theplatform with Nilotinib (FIG. 77C), and Tg-APP+Nilotinib entered SE(platform entry, clear bars) significantly more times than did controlmice (FIG. 77D, 30% higher than control), while Tg-APP+DMSO didsignificantly worse than control (FIG. 77D, 25%). The distance traveled(FIG. 77D, black bars) was also significantly reduced in DMSO (86%)compared to Nilotinib treated Tg-APP mice, which had values 30% abovecontrol levels (FIG. 77D, P<0.05, N=12). Novel object recognition wasalso tested and showed that Tg-APP+Nilotinib performed significantlybetter at finding new objects (FIG. 77E, 31%, p<0.001, N=17) than DMSOmice, while Aβ₁₋₄₂ parkin−/− mice did not learn with or withoutNilotinib (FIG. 77E, N=5).

Example 6

These studies shows that parkin ubiquitinates TDP-43 and facilitates itscytosolic accumulation through a multi-protein complex with HDAC6.

Experimental Procedures.

Stereotaxic Injection—

Stereotaxic surgery was performed to inject the lentiviral (Lv)constructs encoding either LacZ, parkin and/or TDP-43 into the primarymotor cortex of two-month-old male Sprague-Dawley rats weighing between170-220 g. Animals were injected into left side of the motor cortex with2×10⁹ m.o.i Lv-LacZ and into the right side with 1×10⁹ m.o.iLv-parkin+1×10⁹ m.o.i Lv-LacZ; or 1×10⁹ m.o.i Lv-TDP-43+1×10⁹ m.o.iLv-LacZ; or 1×10⁹ m.o.i Lv-parkin+1×10⁹ m.o.i Lv-TDP-43. All animalswere sacrificed two weeks post-injection and the left cortex wascompared to the right cortex. A total of 8 animals of each treatment (32animals) were used for WB, ELISA and immuno-precipitation and 8 animalsof each treatment (32 animals) for immunohistochemistry. A total N=64animals were used. Transgenic hemizygous mice harboring human TDP-43with the A315T mutation under the control of prion promoter and C57BL6/Jmice controls were used. The colony was obtained from Jackson LaboratoryRepository (JAX Stock No. 010700) and displayed a lifespan considerablyshorter than previous reports, with almost 90% of all pups, includingmales and females manifesting motor symptoms around 21-30 days.Hemizygous mice were bred via mating of hemizygous with non-carrier wildtype C57BL/6, and upon genotyping, half were identified as transgenicand the other half was non-transgenic control. All mice used are F1generation from direct mating between hemizygous and C57BL/6 mice. Thesestudies were approved and conducted according to Georgetown UniversityAnimal Care and Use Committee (GUACAC).

Cell Culture and Transfection.

Human neuroblastoma M17 cells (seeding density 2×10⁵ cells) were grownin 24 well dishes (Falcon) to 70% confluence in Dulbecco's ModifiedEagle Medium (DMEM; Invitrogen) plus 10% (v/v) heat-inactivated fetalbovine serum (Invitrogen), penicillin/streptomycin, and 2 mM L-glutamineat 37° C. and 5% CO2, washed twice in phosphate-buffered saline (PBS).Transient transfection was performed with 3 μg parkin cDNA or 3 μgTDP-43 cDNA, or 3 μg LacZ cDNA. Cells were treated with 5 μM tubacin for24 hours and DAPI stained in 12 well dishes. Cells were harvested 24hours after transfection. Transfection was performed in DMEM withoutserum using Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocol. Cells were harvested one time with lysis buffer(20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid,1 mM ethyleneglycoltetraacetic acid (EGTA), 1% Triton X-100, 2.5 mMsodium pyrophosphate, 1 mM 3-glycerophosphate, 1 mM sodiumorthovanadate, 1 μg/ml leupeptin, and 0.1 mM PMSF) and centrifuged at10,000×g for 20 min at 4° C., and the supernatant was collected. Westernblot was performed on NuPAGE 4-12% Bis-Tris gel (Invitrogen). Proteinestimation was performed using the microscale BioRad protein assay(BioRad Laboratories Inc, Hercules, Calif., USA).

Western Blot Analysis—

The cortex was dissected out and homogenized in 1×STEN buffer (50 mMTris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSFand protease cocktail inhibitor). The pellet was then re-suspended in 4Murea and homogenized and centrifuged at 5.000 g and the supernatantcontaining the insoluble protein fraction was collected. Total TDP-43was probed either with (1:1000) mouse monoclonal (2E2-D3) antibodygenerated against N-terminal 261 amino acids of the full length protein(Abnova) or (1:1000) Rabbit polyclonal (ALS10) antibody (ProteinTech,Cat #10782-2-AP). Rabbit polyclonal anti-ubiquitin (ChemiconInternational) was used (1:1000), and rabbit polyclonal anti-parkin(Millipore) antibody was used (1:1000) for WB. Rabbit polyclonalanti-actin (Thermo Scientific) was used (1:1000). Rabbit polyclonalanti-SQSTM1/p62 (Cell Signaling Technology) was used (1:500). Rabbitmonoclonal (1:1000) HDAC6 (Cell Signaling Technology) was used. SIAH2was probed (1:400) with mouse monoclonal antibody (Novus Biologicals)and HIF-1α with (1:1000) mouse monoclonal antibody (Novus Biologicals).Immuno-precipitation was performed on a total of 100 mg protein with(1:100) rabbit polyclonal TDP-43 antibody (ProteinTech), or rabbitmonoclonal (1:100) parkin antibody (Invitrogen) and then compared withthe input samples. Western blots were quantified by densitometry usingQuantity One 4.6.3 software (Bio Rad). Densitometry was obtained asarbitrary numbers measuring band intensity. Data were analyzed asmean±St.Dev, using ANOVA, with Neumann Keuls multiple comparison betweentreatment groups.

Parkin Enzyme-Linked Immunosorbent Assay (ELISA)—

was performed on brain soluble brain lysates (in STEN buffer) orinsoluble brain lysates (4M urea) using mouse specific parkin kit(MYBioSource) in 50 μl (1 μg/μl) of brain lysates detected with 50 μlprimary antibody (3 h) and 100 μl anti-rabbit antibody (30 min) at RT.Extracts were incubated with stabilized Chromogen for 30 minutes at RTand solution was stopped and read at 45 0 nm, according tomanufacturer's protocol.

Parkin E3 Ubiquitin Ligase Activity.

To determine the activity of parkin E3 ligase activity E3LITECustomizable Ubiquitin Ligase Kit (Life Sensors, UC #101), whichmeasures the mechanisms of E1-E2-E3 activity in the presence ofdifferent ubiquitin chain, was used. To measure parkin activity in thepresence or absence of substrates, parkin (1:100) was immunoprecipitatedwith PRK8 antibodies and TDP-43 (1:100) was immunoprecipitated withhuman TDP-43 (Abnova) from 100 mg TDP43-Tg brain lysates. UbcH7 was usedas an E2 that provides maximum activity with parkin E3 ligase and addedE1 and E2 in the presence of recombinant ubiquitin, including wild typecontaining all seven possible surface lysines, no lysine mutant (K0), orK48 or K63 to determine the lysine-linked type of ubiquitin. E3 wasadded as IP parkin or recombinant parkin (Novus Biologicals) to an ELISAmicroplate that captures polyubiquitin chains formed in the E3-dependentreaction, which was initiated with ATP at room temperature for 60minutes. Also included were an E1-E2-E3 and a polyubiquitin chaincontrol in addition to E1, E2 and TDP-43 without parkin and assay bufferfor background reading. The plates were washed 3 times and incubatedwith detection reagent and streptavidin-HRP for 5 minutes and thepolyubiquitin chains generated by E1-E2-E3 machinery were read on achemiluminecense plate reader.

Immunoprecipitation and Ubiquitination Assay.

Either TDP-43 or parkin were separately immunoprecipitated in 100 μl(100 μg of proteins) 1×STEN buffer using (1:100) human specificanti-TDP-43 monoclonal antibody (Abnova) or (1:100) anti-parkin mousemonoclonal antibody (PRK8; Signet Labs; Dedham, Mass.), respectively.Following immunoprecipitation, 300 ng of each substrate protein (parkinand TDP-43) were mixed in the presence of 1 μg recombinant humanubiquitin (Boston Biochem, Mass.), 100 mm ATP, 1 μg recombinant UbcH7(Boston Biochem), 40 ng E1 recombinant enzyme (Boston Biochem) andincubated at 37° C. in an incubator for 20 min. The reaction was heatinactivated by boiling for 5 min and the substrates were analyzed bywestern blot.

Immunohistology—

Immunohistochemistry was performed on 20 μm-thick sections of brain orcervical spinal cord. TDP-43 was probed (1:200) with rabbit polyclonal(ALS10) antibody (ProteinTech, Cat #10782-2-AP). Rabbit polyclonalanti-ubiquitin (Chemicon International) was used (1:100), and mousemonoclonal anti-parkin (Millipore) antibody was used (1:200) forimmunohistochemistry. Toluidine blue and DAPI staining were performedaccording to manufacturer's instructions (Sigma). Counting of Toluidineblue staining of centric axons within 10 random fields of each slide wasperformed by a blind investigator in N=8 animals of each treatment. Allstaining experiments were scored by a blind investigator to thetreatments.

20S Proteasome Activity Assay—

Brain extracts 100 μg were incubated with 250 μM of the fluorescent 20Sproteasome specific substrate Succinyl-LLVY-AMC at 37° C. for 2 h. Themedium was discarded and homogenates were lysed in 50 mM HEPES, pH 7.5,5 mM EDTA, 150 mM NaCl and 1% Triton X-100, containing 2 mM ATP. Thefluoropore 7-Amino-4-methylcoumarin (AMC), which is released aftercleavage from the labeled substrate Succinyl-LLVY-AMC (ChemiconInternational, Inc.), is detected and free AMC fluorescence isquantified using a 380/460 nm filter set in a fluorometer (absorption at351 nm and emission at 430 nm). Non-proteasomal side reactivity wasmeasured by adding lactacystin as a specific proteasome inhibitor to thereaction mix and subtracted these values from total for an accuratemeasure of specific proteasome activity.

qRT-PCR in Neuronal Tissues.

qRT-PCR was performed on Real-time OCR system (Applied Biosystems) withFast SYBR-Green PCR master Mix (Applied Biosystems) in triplicate fromreverse transcribed cDNA from control un-injected, or lentiviral LacZ,parkin, TDP-43 and TDP-43+parkin injected rat cortical brain tissues.These experiments were repeated in human neuroblastoma M17 cells andA315T-Tg compared to non-transgenic C57BL/6 controls. Human wild-typeparkin forward primer CCA TGA TAG TGT TTG TCA GGT TC and a reverseprimer GTT GTA CTT TCT CTT CTG CGT AGT GT were used. Gene expressionvalues were normalized using GADPH levels.

Results

TDP-43 Inhibits Proteasome Activity and Alters Parkin Protein Levels.

To determine the effects of TDP-43 on parkin in transgenic animals, theA315T mutant TDP-43 transgenic mice (TDP43-Tg), which were reported tohave aggregates of ubiquitinated proteins in layer 5 pyramidal neuronsin frontal cortex, as well as spinal motor neurons, without cytoplasmicTDP-43, was used. This model is relevant to these studies because itshows nuclear TDP-43 driven pathology, independent of cytoplasmic TDP-43inclusions. Western blot analysis showed accumulation of full length andTDP-43 fragments (˜35 kDA) as well as higher molecular weight specieswith human TDP-43 antibody (FIG. 69A, 1st blot) compared tonon-transgenic controls, suggesting TDP-43 pathology. Further analysisof the soluble brain lysate (STEN extract) showed increased parkinlevels by Western blot (FIG. 69A, 2nd blot, 82%, P<0.05, N=8) andappearance of a lower molecular weight band, perhaps indicating parkincleavage. Increased levels of ubiquitin smears (FIG. 69A, 3rd blot) werealso observed using anti-ubiquitin antibodies, suggesting accumulationof ubiquitinated proteins. It was determined whether parkin solubilitywas altered in TDP43-Tg mice. The protein pellet was resuspended afterSTEN extraction in 4M urea to detect the insoluble fraction and wedetected a significant increase (FIG. 69B, 95% by densitometry, P<0.05,N=8) in insoluble parkin in 1-month old TDP43-Tg mice compared tocontrol (FIGS. 69B&C, P<0.05, N=8), suggesting that TDP-43 aggregatesare associated with altered parkin solubility. The ratio of soluble overinsoluble parkin was not significantly changed (FIG. 69C, P<0.05),suggesting that TDP-43 accumulation increases soluble and insolubleparkin levels. Probing for TDP-43 in 4M urea extracts was also performedand increased levels of insoluble TDP-43 (FIG. 69B, 2nd blot) weredetected in TDP43-Tg compared to control. To verify the changes inparkin level observed by WB, quantitative parkin ELISA was performed todetermine the levels of both soluble (STEN extract) and insoluble (4Murea) parkin, using brain extracts from parkin^(−/−) mice as control forELISA specificity (FIG. 69D, N=8). A significant increase in bothsoluble (46%, P<0.05) and insoluble (64%) parkin was detected inTDP43-Tg mice compared to control level (FIG. 69D, P<0.05, N=8), furthersuggesting an increase in parkin level and insolubility in TDP43-Tgmice.

The seven in absentia homolog (SIAH) protein is another E3 ligaseinvolved in ubiquitination and proteasomal degradation of specificproteins. SIAH is rapidly degraded via the proteasome. SIAH2 was used asan E3 ligase control to determine whether TDP-43 decreases parkinsolubility, leading to alteration of its E3 ligase function independentof other E3 ligases. Western blot analysis showed a significant increase(215%) in soluble SIAH2 levels (FIGS. 69E&F, P<0.05, N=8) in TDP43-Tgmice compared to control, indicating lack of degradation of SIAH2perhaps due to proteasomal impairment. However, SIAH2 was not detectedin the insoluble fraction. A lower molecular weight band was alsoobserved at 17 kDa (FIG. 69E) in transgenic mice, suggesting possiblecleavage of SIAH2 dimeric structure. Further examination of the level ofSIAH2 target molecule HIF-1α showed a significant increase (76%, P<0.05)in protein level (FIGS. 69E&F), suggesting lack of proteasomaldegradation.

To ascertain the effect of TDP-43 on parkin level and proteasomeactivity wild type TDP-43, (FIG. 69G, 1st blot) was expressed in thepresence or absence of parkin (FIG. 69G, 2nd blot) in human M17neuroblastoma cells. Expression of TDP-43 alone led to appearance ofendogenous parkin protein (FIG. 69G, 2nd blot), suggesting that TDP-43regulates parkin mRNA to induce protein expression. Co-expression ofexogenous parkin and TDP-43 led to a slight decrease in TDP-43 levels(FIG. 69G, 1st blot) and a noticeable decrease in ubiquitinated proteins(FIG. 69G, 3rd blot) compared to TDP-43 alone. SIAH2 was difficult todetect in control M17 cells (FIG. 69G, 4th blot), but accumulated whenTDP-43 was expressed despite the increase in endogenous parkin, however,exogenous parkin co-expression with TDP-43 led to disappearance of SIAH2(FIG. 69G, 4th blot). The effects of parkin expression alone (FIG. 69G,2nd blot) were further compared to LacZ on TDP-43 and SIAH2 levels. Nodifferences were observed between control (FIG. 69F), LacZ and parkintransfected M17 cells (FIG. 69H) on endogenous TDP-43 expression level(FIG. 69H, 1st blot). A higher level of ubiquitinated protein smearswere observed with parkin expression (FIG. 69H, 3^(rd) blot), consistentwith parkin role as an E3 ubiquitin ligase, but the level of SIAH2 wassignificantly decreased (FIG. 69H, 4th blot, 74%, P<0.05) compared toactin control. To determine whether SIAH2 accumulation is due todecreased E3 ligase activity or proteasomal function, we measuredproteasome activity (FIG. 69I) and found that TDP-43 significantlydecreased (66%) proteasome activity (P<0.05, N=12), while parkinco-expression significantly reversed proteasome activity to 74% ofcontrol or parkin levels, but remained significantly less (26%) thancontrol. These data show that TDP-43 increases parkin expression levels,while proteasomal inhibition leads to decreased degradation of proteins,including the rapidly degrading SIAH2.

Lentiviral Expression of TDP-43 in Rat Motor Cortex Results in IncreasedProtein Levels in Preganglionic Cervical Spinal Cord Inter-Neurons.

Wild type TDP-43 was expressed using lentiviral gene delivery into themotor cortex of 2-month old Sprague Dawley rats. Immunohistochemistryusing rabbit polyclonal antibody that recognizes human and rat TDP-43(ALS10, ProteinTech) showed increased TDP-43 protein levels andcytosolic accumulation 2 weeks post-injection (FIG. 70B) compared toLacZ injected contralateral (FIG. 70A) hemisphere. To ascertainspecificity of gene expression, human specific (hTDP-43) mousemonoclonal antibody that recognizes a.a.1-261 (Abcam) was used andpositive human TDP-43 staining was observed within 4 mm radius in 38%(by stereology, N=8) of cortical neurons (FIG. 70D) compared to LacZinjected (FIG. 82C) hemisphere. Further examination of cervical spinalcord revealed 13% increase in immunoreactivity to hTDP-43 (FIG. 70F) andincreased reactivity to TDP-43 antibody (FIG. 70G) in preganglionicinter-neurons, which were morphologically identified in thecontralateral side of TDP-43 injected motor cortex (FIG. 70E) comparedto the contralateral spinal cord injected with LacZ (FIGS. 70H&I),suggesting that hTDP-43 expression in the motor cortex leads toincreased protein levels in the contralateral spinal cord. Furthermore,stereological counting revealed 46% (by stereology, N=8) increase in thelevels of hTDP-43 (FIG. 70J) and increased immunoreactivity to TDP-43antibody (FIG. 70K) in the dorso-cortical spinal tract (DCST) ofcervical spinal cord contralateral to cortical TDP-43 expressioncompared to LacZ injected side (FIGS. 70L&M). Toluidine blue stainingand quantification by a blind investigator of centric axons within 10random fields of each slide showed increased number (18%, N=8) of axons(FIG. 70N, arrows) in enlarged circles, suggesting axonal degenerationcompared to the contralateral DCST (FIG. 70O). Some centric axons weredetected in all treatments.

Lentiviral Parkin Expression Increases Cytosolic Co-Localization ofTDP-43 with Ubiquitin.

Because TDP-43 is detected in ubiquitinated forms within the cytosol inhuman disease, it was sought to determine whether ubiquitination isbeneficial or detrimental to TDP-43 using parkin as a ubiquitousE3-ubiquitin ligase in the human brain. TDP-43 was co-expressed withparkin and animals were sacrificed 2 weeks post-injection. Staining of20 μm thick brain sections showed endogenous parkin expression (FIG.71A) and TDP-43 (FIG. 71B), which was predominantly localized to DAPIstained nuclei (FIG. 71C) in LacZ-injected rat motor cortex. Stainingwith anti-ubiquitin antibodies (FIG. 71D) in rats expressing TDP-43 inthe motor cortex (FIG. 71E) did not result in any noticeableco-localization between TDP-43 and ubiquitin (FIG. 71F). Stereologicalcounting showed 38% increase in hTDP-43 stained cells (FIG. 71D).However, cytosolic TDP-43 was observed in cortical neurons expressingTDP-43 (FIG. 71F) compared to nuclear TDP-43 in LacZ injected animals(FIG. 71C). We expressed parkin in the rat motor cortex (FIG. 71G)together with TDP-43 (FIG. 71H) and observed cytosolic co-localizationof parkin and TDP-43 (FIG. 71I, 35% by stereology). We further stainedwith anti-ubiquitin antibodies and observed increased levels ofubiquitin (FIG. 71J, 35% by stereology) in animals injected with parkinand TDP-43 (FIG. 71K). Interestingly, enhanced ubiquitin signalsco-localized with TDP-43 in the cytosol, suggesting that ubiquitinationmay result in cytosolic sequestration of TDP-43. To determine whetherexogenous parkin expression affects endogenous TDP-43 proteinlocalization, we stained with parkin (FIG. 71M, 28% by stereology) andTDP-43 (FIG. 71N) antibodies, but we did not observe any changes in thepattern of TDP-43 staining (FIG. 71O).

Parkin Promotes K48 and K63-Linked Ubiquitin to TDP-43.

To demonstrate whether parkin mediates TDP-43 ubiquitinationimmuno-precipitation was performed to show ubiquitinated TDP-43 in thepresence of parkin expression. Western blot analysis of the input showedthat increased exogenous parkin (FIG. 72A, 1st blot, N=8, P<0.05, 42%)in the rat motor cortex, increases the levels of ubiquitinated proteins(FIG. 72A, 2nd blot). Densitometry analysis of TDP-43 blots (FIG. 72A,3rd blot) showed a significant increase (48%, N=8) in TDP-43 levels inbrains injected with lentiviral TDP-43 (consistent with our previouswork compared to LacZ or parkin injected brains. However, co-injectionof TDP-43 and parkin did not result in any significant changes in TDP-43levels (P<0.05, N=8), suggesting that parkin mediates TPD-43ubiquitination, which may not lead to protein degradation. Anon-functional parkin mutant (T240R, threonine to arginine mutation),which was co-expressed with TDP-43 (FIG. 72A, top blot) was also usedand no changes in ubiquitinated proteins (FIG. 72A, 2nd blot) or TDP-43levels (FIG. 72A, 3rd blot) were detected. TDP-43 wasimmune-precipitated and probed with ubiquitin (FIG. 72A, 4th blot) toascertain that high molecular weight species are ubiquitinated TDP-43proteins and not some protein aggregates. An increase in protein smearwas observed when TDP-43 was co-injected with parkin, compared toTDP-43, parkin or LacZ alone, suggesting increased TDP-43 ubiquitinationin the presence of wild type parkin. However, no differences wereobserved in the levels of ubiquitinated proteins (FIG. 72A, 4th blot)when TDP-43 was immuno-precipitated with or without expression of T240Rmutant parkin, suggesting that functional parkin mediates TDP-43ubiquitination.

To determine whether TDP-43 affects parkin E3 ubiquitin ligase activity,parkin (FIG. 72B, left blot) and TDP-43 (FIG. 4B, right blot) wereimmune-precipitated and an enzyme activity assay was performed. Positivecontrols with E1-E2-E3 or poly-ubiquitin chains or recombinant parkin(Novus Biologicals) were used to measure E3 ubiquitin ligase activityand poly-ubiquitin chain readings (FIG. 72C). No parkin activity wasdetected with the lysine null (K0) ubiquitin, but either mutant K48 orK63-linked ubiquitin showed an increase in parkin E3 ubiquitin ligaseactivity compared to control K0 (FIG. 72C, N=4). Parkin activity withK63 ubiquitin was significantly higher (83%, P<0.05, N=4) thanK48-linked ubiquitin, suggesting that parkin undergoes K48 andK63-linked auto-ubiquitination. Parkin was also ubiquitinated using wildtype ubiquitin, which contains all 7 lysine residues. To determinewhether parkin activity is altered in the presence of TDP-43, bothparkin and TDP-43 were added to the enzyme mix. As expected no activitywas detected with lysine null ubiquitin (K0), but parkin activity wassignificantly increased compared to parkin alone (FIG. 72C, P<0.05, N=8)with K48 (154%) and K63 (156%) ubiquitin, indicating that parkinactivity is even higher in the presence of a substrate. Parkin alsoshowed a significantly higher level of activity with wild type ubiquitinin the presence of TDP-43 (279%) compared to parkin alone.

To ascertain that parkin mediates ubiquitination of TDP-43, parkin andTDP-43 were immunoprecipitated separately and in vitro ubiquitinationassays were performed. Incubation of both parkin and TDP-43 in thepresence of either wild type (FIG. 72D, 2nd lane) or K48 (7th lane) orK63 (8th lane) ubiquitin (FIG. 72D, N=3), showed a protein smear upon WBanalysis with TDP-43 antibodies compared to lysine null (K0) ubiquitin(6th lane), or in the absence of E1 or E2 or both (all other lanes,suggesting that parkin mediates K48 and K63-linked ubiquitination ofTDP-43. Additionally, parkin incubation in the presence of either wildtype (FIG. 72E, 2nd lane) or K48 (7th lane) or K63 (8th lane) ubiquitin(FIG. 72E, N=3), showed a protein smear upon WB analysis with parkinantibodies compared to lysine null (K0) ubiquitin (6th lane), or in theabsence of E1 or E2 or both (all other lanes, suggesting that parkinundergoes K48 and K63-linked auto-ubiquitination.

The activity of the 20S proteasome (FIG. 72F), which was significantlydecreased (31%, P<0.05) when TDP-43 was expressed alone (N=8, P<0.05),but co-expression of parkin significantly reversed proteasome activity(48%, P<0.05) compared to TDP-43 alone, was measured. However,proteasome activity in parkin expressing cortex remained significantlyhigher than LacZ (73%, P<0.05) and parkin+TDP-43 (31%, P<0.05) injectedanimals, indicating that parkin activity partially reverses proteasomeactivity.

Parkin Forms a Multi-Protein Complex with HDAC6 to Mediate TDP-43Translocation from Nucleus to Cytosol.

Lack of degradation of ubiquitinated TDP-43 and cytosolic accumulationof parkin, TDP-43 and ubiquitin in gene transfer animals led toexamination of possible mechanisms to translocate TDP-43 to the cytosol.Western blot analysis showed a significant increase (41%, P<0.05) inHDAC6 levels when TDP-43 was expressed compared to LacZ or parkininjected animals (FIGS. 72G&H, 1st blot, P<0.05, N=8). However, furtherincreases in HDAC6 levels (FIGS. 72G&H, 112%, P<0.05) were detected whenparkin was co-expressed with TDP-43, suggesting possible interactionbetween these proteins. Examination of molecular markers of autophagyshowed a significant increase in P62 (28%, P<0.05) when parkin wasco-expressed with TDP43 (FIGS. 72G&H, 2nd blot) compared to all othertreatments, suggesting accumulation of ubiquitinated proteins. Nochanges in other markers of autophagy (LC3, beclin, Atgs) or appearanceof autophagic vacuoles by EM were seen. Human TDP-43 wasimmunoprecipitated from transgenic mice and TDP-43 was verified at 46kDa using hTDP-43 antibody (FIG. 73A, 1st & 2nd blots). Stripping andre-probing with parkin antibody showed a slightly higher band around 50kDa, suggesting presence of parkin protein (FIG. 73A, 3rd blot). Furtherstripping and probing with HDAC6 antibody (FIG. 73A, 4th blot) showed ahigher molecular weight band around 120 kDa, indicating a multi-proteincomplex between parkin, TDP43 and HDAC6. A reserve experiment wasperformed via parkin immuno-precipitation and verification of humanTDP-43 presence (FIG. 73B, 1st & 2nd blot). Stripping and probing withparkin antibody showed parkin band in both transgenic and non-transgeniccontrol mice (FIG. 73B, 3rd blot), indicating that parkin wassuccessfully immuno-precipitated. A higher molecular weight bandrepresentative of HDAC6 (FIG. 73B, 4th blot) was detected in transgenicbut not control mice, further suggesting multi-protein complex formationbetween TDP43, parkin and HDAC6.

To ascertain that both parkin and HDAC6 are required for TDP-43translocation, GFP-tagged TDP-43 was expressed in M17 neuroblastomacells in the presence of wild type or loss-of-function mutant (T240R)parkin, and treated with 5 μM selective HDAC6 inhibitor for 24 hours.GFP expression was predominantly observed within DAPI-stained nuclei inlive M17 cells (FIG. 73C, insert is higher magnification), howeverparkin co-expression led to significant GFP fluorescence within thecytoplasm (FIGS. 73D&E) and neuronal processes (FIG. 73D, insert showshigher magnification of GFP fluorescence). Treatment with the HDAC6inhibitor, tubacin, did not lead to GFP fluorescence in the cytosol inthe presence (FIG. 73F) or absence (FIG. 73G) of parkin. Loss of parkinE3 ubiquitin ligase function (T240R) did not lead to TDP-43 accumulationin the cytosol (FIG. 73H), suggesting that the E3 ubiquitin ligasefunction of parkin and HDAC6 activity are required to facilitate TDP-43accumulation within the cytosol.

To verify whether TDP-43 expression increases parkin mRNA levels,performed qRT-PCR was performed in samples isolated from rat cortex,human M17 cells and TDP43-Tg mice. Park2 mRNA levels in M17 cellsexpressing parkin was significantly higher (FIGS. 73I&J, 55%, P<0.05,N=4) than LacZ, but similar to TDP-43 injected brains (61%, P<0.05).Parkin co-expression with TDP-43 showed significantly higher levels ofpark2 mRNA (FIG. 73J, 74%, P<0.05, N=4) compared to parkin alone.Similarly, Park2 mRNA levels in rat brains expressing parkin wassignificantly higher (FIGS. 73K&L, 41%, P<0.05, N=4) than LacZ animals,as well as TDP-43 injected brains (21%, P<0.05). However, parkinco-expression with TDP-43 showed significantly higher levels of park2mRNA (FIG. 73J, 84%, P<0.05, N=4) compared to all other treatments.Therefore, park2 mRNA levels between TDP43-Tg and non-transgenic controllittermates were compared. A significant increase (FIGS. 73M&N, 114%,N=4, P<0.05) in park2 mRNA was observed in TDP43-Tg brains injectedcompared to C57BL/6 controls, showing that parkin is a transcriptionaltarget for TDP-43.

Example 7

Parkin Plays an Essential Role in Motor Neuron Survival Via Modulationof Nuclear TDP-43 Transport to the Synapse

E3 ubiquitin ligase Parkin is important in neurodegeneration. Parkinpromotes specific ubiquitination of TAR-DNA binding protein (TDP)-43,and could mediate its transport via complex formation with histonedeacetylase 6 (HDAC6). In healthy neurons, TDP-43 is predominantlynuclear and could be transported to the synapse for generation ofsynaptic proteins. As shown in FIG. 74, 1). Parkin could ubiquitinateTDP-43 and translocate it from the nucleus to the cytosol; 2).Parkin-HDAC6 complex is required for axoplasmic TDP-43 transport to thesynapse; and 3). TDP-43 availability at the synapse modulates expressionof synaptic proteins that maintain glutamate metabolism.

Long motor neurons, which degenerate in Amyotrophic Lateral Sclerosis(ALS), could depend on axonal TDP-43 transport to distant synapses, thusincreasing their vulnerability to TDP-43 localization. Inneurodegeneration, including ALS and Frontotemporal Dementia (FTD-TDP),wild type and mutated TDP-43 aggregate, and neurons bearing TDP-43aggregates express less parkin. Data provided herein show that parkinalters TDP-43 localization, reverses TDP-43-induced alteration inglutamate levels and improves motor performance. TDP-43 binds to mRNAsthat code for proteins involved in synaptic function, includingsynaptotagmin and vesicular glutamate transporters. Glutamate transportis defective in ALS, due to loss of glutamate transporters thatfacilitate conversion of synaptic glutamate into glutamine. Thus,nuclear TDP-43 translocation and axoplasmic transport to the synapsecould be particularly important for motor neurons.

Parkin-mediated TDP-43 localization to the synapse could affect synapticproteins that maintain glutamate metabolism. Thus, parkin could play anessential role in motor neuron survival via modulation of nuclear TDP-43transport to the synapse.

The following data support these conclusions. FIG. 75 shows thedistribution of GFP-tagged TDP-43 in M17 cells transfected with 3 mgcDNA for 24 hrs and then treated with Nilotinib (10 mM) or Bosutinib (5mM) and HDAC6 inhibitor Tubacin (5 mM) for additional 24 hrs. Inserts(B&D) represent higher magnification images showing translocation ofGFP-tagged TDP-43 from nucleus (A) into the cytosol (B&D, and inserts),while tubacin impairs translocation (C&E).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method of treating a neurodegenerative diseasein a subject in need thereof, comprising: selecting a subject with aneurodegenerative disease of the central nervous system or at risk for aneurodegenerative disease of the central nervous system; andsystemically administering to the subject an effective amount of atyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor crossesthe blood brain barrier, wherein the effective amount of the tyrosinekinase inhibitor is less than 10 mg/kg, and wherein the effective amountof the tyrosine kinase inhibitor is lower than a chemotherapeuticdosage, wherein the tyrosine kinase inhibitor is selected from the groupconsisting of nilotinib, bosutinib, and a combination thereof.
 2. Themethod of claim 1, wherein the neurodegenerative disease is selectedfrom the group consisting of Amyotrophic Lateral Sclerosis, Alzheimer'sDisease, Parkinson's Disease, Huntington's Disease, and Mild CognitiveImpairment, an α-Synucleinopathy or a Tauopathy.
 3. The method of claim1, wherein the effective amount of the tyrosine kinase inhibitorpromotes Parkin activity.
 4. The method of claim 1, wherein the tyrosinekinase inhibitor is administered daily.
 5. The method of claim 1,further comprising administering a second therapeutic agent to thesubject.
 6. A method of inhibiting toxic protein aggregation in a neuronof a subject in need thereof with a neurodegenerative disorder,comprising contacting the neuron in the subject with an effective amountof a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitorcrosses the blood brain barrier, wherein the neuron is contacted withthe tyrosine kinase inhibitor by systemically administering a tyrosinekinase inhibitor to the subject at a dosage of less than 10 mg/kg, andwherein the effective amount of the tyrosine kinase inhibitor is lowerthan a chemotherapeutic dosage, wherein the tyrosine kinase inhibitor isselected from the group consisting of nilotinib, bosutinib, and acombination thereof.
 7. The method of claim 6, wherein the protein isselected from the group consisting of an amyloidogenic protein,alpha-synuclein, tau, insoluble Parkin, or TDP-43.
 8. The method ofclaim 7, wherein the amyloidogenic protein is β-amyloid.
 9. A method ofrescuing a neuron from neurodegeneration associated with aneurodegenerative disorder in a subject in need thereof comprisingcontacting the neuron in the subject with an effective amount of atyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor crossesthe blood brain barrier, wherein the neuron is contacted with thetyrosine kinase inhibitor by systemically administering a tyrosinekinase inhibitor to the subject at a dosage of less than 10 mg/kg, andwherein the effective amount of the tyrosine kinase inhibitor is lowerthan a chemotherapeutic dosage wherein the tyrosine kinase inhibitor isselected from the group consisting of nilotinib, bosutinib, and acombination thereof.
 10. The method of claim 1, further comprisingdetermining that the subject has a decreased level of parkin activityrelative to a control prior to administering to the subject an effectiveamount of the tyrosine kinase inhibitor.
 11. A method of treating anα-Synucleinopathy in a subject in need thereof, comprising selecting asubject with an α-Synucleinopathy or at risk for an α-Synucleinopathyand systemically administering to the subject an effective amount ofbosutinib, wherein the bosutinib is administered to the subject at adosage of about 5 mg/kg or less.
 12. The method of claim 11, wherein theeffective amount of bosutinib promotes Parkin activity.
 13. The methodof claim 11, wherein the bosutinib is administered daily.
 14. The methodof claim 11, further comprising administering a second therapeutic agentto the subject.
 15. A method of inhibiting toxic protein aggregation ina neuron of a subject in need thereof with an α-Synucleinopathy,comprising contacting the neuron in the subject with an effective amountof bosutinib, wherein the neuron is contacted with bosutinib bysystemically administering bosutinib to the subject at a dosage of about5 mg/kg or less.
 16. The method of claim 15, wherein the protein isselected from the group consisting of a-synuclein, and insoluble Parkin.17. A method of rescuing a neuron from neurodegeneration associated withan α-Synucleinopathy in a subject in need thereof comprising contactingthe neuron in the subject with an effective amount of bosutinib, whereinthe neuron is contacted with the bosutinib by systemically administeringa bosutinib to the subject at a dosage of about 5 mg/kg or less.